Lipid vesicle compositions and methods of use

ABSTRACT

The invention provides delivery systems comprised of stabilized multilamellar vesicles, as well as compositions, methods of synthesis, and methods of use thereof. The stabilized multilamellar vesicles may comprise prophylactic, therapeutic and/or diagnostic agents.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 61/315,485, entitled “MICRO- ANDNANOPARTICLES WITH SELF-ASSEMBLED LIPID COATINGS FOR SURFACE DISPLAY OFDRUGS SUCH AS VACCINE ANTIGENS AND ADJUVANTS” filed on Mar. 19, 2010,and U.S. Provisional Application Ser. No. 61/319,709, entitled“INTERBILAYER CROSSLINKED MULTILAMELLAR VESICLES” filed on Mar. 31,2010, both of which are incorporated by reference herein in theirentireties.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. CA140476from the NIH, and Grant No. W911NF-07-D-0004 from the Department ofDefense. The Government has certain rights in the invention.

BACKGROUND OF INVENTION

Liposomes have been widely used as a delivery vehicle for smallmolecules; however, it remains difficult to achieve high levels ofencapsulation for many macromolecular drugs within liposomes and manydrug formulations leak from liposomes too quickly to maintain usefuldrug delivery kinetics. While drug delivery by micro- and nanoparticlescan encapsulate proteins and small-molecule drugs, this still typicallyyields very low total mass encapsulated drug per mass of particles,typically on the order of ˜10 μg drug/mg particles. In addition, theorganic solvents used in polymer particle synthesis andhydrophobic/acidic environment within these particles can lead todestruction of therapeutics. (See Zhu et al. Nat. Biotechnol. 200018:52-57.)

SUMMARY OF INVENTION

The invention provides novel and inventive drug delivery systems withhigher loading capability, a capacity to sequester high levels of bothhydrophobic and hydrophilic agents simultaneously, and longer releaseprofiles. Some aspects of these delivery systems comprise stabilizedmultilamellar lipid vesicles having crosslinked lipid bilayers (referredto herein as interbilayer-crosslinked multilamellar vesicles or ICMV).

As shown in the Examples, the vesicles of the invention have unexpectedenhanced encapsulation efficiency (e.g., in some instances, 100-foldmore efficient than simple liposomes), and they are able to releaseencapsulated (or otherwise entrapped) agents via slow and sustainedkinetics even in the presence of serum, making them highly desirable assustained delivery vehicles in vivo. Moreover, as described in greaterdetail herein, the vesicles of the invention may be synthesized inaqueous environments, thereby avoiding the harsh conditions that arecommon in various prior art methods including the use of organicsolvents and/or acidic environments. As a result, these synthesismethods are more suitable for a variety of agents including those thatwould typically be compromised structurally and/or functionally usingsuch prior art methods. The resultant vesicles are therefore free oforganic solvent and may be comprised solely of the lipids, includingbiodegradable lipids, and any agent encapsulated to therein ortherethrough.

The invention is based in part on these and other surprising andunexpected characteristics of the vesicles of the invention, asdescribed in greater detail herein. Accordingly, the invention providesthese stabilized vesicles, compositions comprising these vesicles,methods of making these vesicles, and methods of use thereof.

Thus, in one aspect, the invention provides a multilamellar lipidvesicle having crosslinks between lipid bilayers, or a plurality orpopulation of multilamellar lipid vesicle having crosslinks betweenlipid bilayers. The plurality may be but is not limited to 1-10³, 1-10⁴,1-10⁵, 1-10⁶, 1-10⁷, 1-10⁸, or 1-10⁹. Various aspects of the vesiclesare described below and it is to be understood that these aspects applyequally to the plurality or population of vesicles, unless otherwisestated. It is to be understood that the vesicles of the inventioncomprise linkages between components of internal adjacent (or apposed)bilayers, and not simply crosslinks between components of the external(or outermost) bilayer or bilayer surface.

In some embodiments, the vesicle comprises a functionalized lipid. Insome embodiments, the functionalized lipid is a maleimide functionalizedlipid. The functionalized lipid may be a phosphoethanolamine but it isnot so limited. In some embodiments, the vesicle comprises a lipidbilayer component that is functionalized, and preferably directlyfunctionalized. In some embodiments, the vesicle comprisesphosphocholine such as but not limited to DOPC. In some embodiments, thevesicle comprises phosphoglycerol such as but not limited to DOPG. Insome embodiments, the vesicle comprises DOPC, DOPG and a functionalizedlipid such as but not limited to a functionalized phosphoethanolamine.The functionalized lipid may be maleimide containing. In someembodiments, the vesicles comprise DOPC, DOPG and a maleimidefunctionalized lipid. The functionalized lipid may be a maleimidefunctionalized phosphoethanolamine such as but not limited to MPB. Insome embodiments, the molar ratio of DOPC:DOPG:functionalized lipid is40:10:50. In some embodiments, the vesicle may comprise DOPC and MPB. Insome embodiments, the vesicles may comprise DOPC, DOTAP and MPB. In someembodiments, the molar ratio of DOPC:DOTAP:functionalized lipid is40:10:50. In some embodiments, the functionalized lipid is present in amolar percentage of at least 25%.

In some embodiments, the vesicle comprises an agent, including one ormore agents. As used herein, one or more agents intends one or moreagents that are different from each other. In some embodiments, theagent is a prophylactic agent, a therapeutic agent, or a diagnosticagent. In some embodiments, the agent is an antigen. In someembodiments, the agent is a protein antigen, including a whole proteinantigen. In some embodiments, the agent is an adjuvant. The adjuvant maybe but is not limited to a TLR agonist such as TLR 4, TLR7, TLR8 andTLR9 agonists. In some embodiments, the vesicles comprise an antigen andat least two adjuvants such as a TLR4 and a TLR7 agonist. In someembodiments, the agent is a protein.

In some embodiments, the vesicles comprise in excess of 300 μg of agentper mg of lipid (or particle), or in excess of 400 μg of agent per mg oflipid (or particle). In some embodiments, the vesicles comprise 300-400μg of agent per mg of lipid (or particle). In some embodiments, thevesicles comprise 325 μg of agent per mg of lipid (or particle), or 407μg of agent per mg of lipid (or particle). In related embodiments, theagent may be protein antigen.

In some embodiments, the agent is encapsulated within the vesicle. Insome embodiments, the agent is present in the core of the vesicle. Insome embodiments, the agent is present between lipid bilayers.

In some embodiments, the vesicles comprise an agent in the core andanother agent in between lipid bilayers. In some embodiments, the agentin the core may be an antigen such as a protein antigen, and the agentin between the lipid bilayers may be an adjuvant such as but not limitedto MPLA. In some embodiments, an antigen is in the core and twoadjuvants are in between the internal lipid bilayers. The two adjuvants,in some embodiments, are MPLA and R-848.

In some embodiments, the vesicles comprise an agent in the core andanother agent in the external bilayer. In some embodiments, the agent inthe core may be an antigen such as a protein antigen, and the agent inthe external bilayer may be an adjuvant such as but not limited to MPLA.In some embodiments, an antigen is in the core and two adjuvants are inthe external bilayer. In some embodiments, an antigen is in the core andone or more adjuvants are in between the internal bilayers and/or on theexternal bilayer surface. The two adjuvants, in some embodiments, areMPLA and R-848.

In some embodiments, the vesicles are conjugated to polyethylene glycol(PEG). In some embodiments, the vesicles may be surface conjugated toPEG.

In another aspect, the invention provides a composition comprising anyof the foregoing multilamellar lipid vesicles (or vesicle populations)and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a composition comprising anyof the foregoing multilamellar lipid vesicles (or vesicle populations)and an excipient suitable for lyophilization. In some embodiments, theexcipient suitable for lyophilization comprises sucrose. The vesiclesmay be used as a stand alone or may be combined with other agents,including any of the agents described herein. In some embodiments, theywill not be administered together with cells and nor will they beconjugated (or otherwise physically attached) to cells, for exampleprior to administration to a subject. The vesicles however may beconjugated to targeting ligands in order to target them to particularcells or sites in the body.

In another aspect, the invention provides a method comprising contactingliposomes comprising a functionalized lipid with a multivalent (e.g.,divalent) cation to form fused liposomes, and contacting the fusedliposomes with a crosslinker to form multilamellar lipid vesicles havingcrosslinks between lipid bilayers, including any of the foregoingvesicles.

In some embodiments, the functionalized lipid is amaleimide-functionalized lipid. In some embodiments, the functionalizedlipid is a functionalized phosphoethanolamine. In some embodiments, thefunctionalized lipid is a maleimide functionalized lipid. In someembodiments, the functionalized lipid is maleimide functionalizedphosphoethanolamine. In some embodiments, the liposomes comprisephosphocholine such as but not limited to DOPC. In some embodiments, theliposomes comprise phosphoglycerol such as but not limited to DOPG. Insome embodiments, the liposomes comprise a maleimide-functionalizedlipid, phosphocholine and phosphoglycerol. In some embodiments, theliposomes comprise DOPC, DOPG and a maleimide functionalized lipid (suchas MPB) at a molar ratio of 40:10:50. In some embodiments, the liposomescomprise DOPC and a maleimide functionalized lipid such as MPB. In someembodiments, the liposomes comprise DOPC, DOTAP and a maleimidefunctionalized lipid (such as MPB), optionally at a molar ratio of40:10:50.

In some embodiments, the linker is a membrane permeable linker. In someembodiments, the crosslinker is a dithiol crosslinker. In someembodiments, the crosslinker is dithiolthrietol (DTT). In someembodiments, dithiol crosslinker to maleimide functionalized lipid molarratio is 1:2.

In some embodiments, the method further comprises conjugatingpolyethylene glycol (PEG) to the surface of the multilamellar lipidvesicles having crosslinks between lipid bilayers.

In some embodiments, the multivalent cations are divalent cations suchas but not limited to Ca²⁺ or Mg²⁺. These may be used alone or incombination.

In some embodiments, the contacting occurs in an aqueous buffer.

In some embodiments, the liposomes comprise an agent. In someembodiments, the agent is a prophylactic agent, a therapeutic agent, ora diagnostic agent.

In another aspect, the invention provides a method comprising contactinga multilamellar lipid vesicle comprising a functionalized lipid bilayercomponent, such as a functionalized lipid, with a crosslinker to formmultilamellar lipid vesicles having crosslinks between lipid bilayers.

In another aspect, the invention provides a method comprisingadministering to a subject a multilamellar lipid vesicle havingcrosslinked lipid bilayers and that comprises an agent, in an effectiveamount. The multilamellar lipid vesicle having crosslinked lipidbilayers may be any of the foregoing multilamellar lipid vesicles havingcrosslinked lipid bilayers or any of those described herein.

In some embodiments, the multilamellar lipid vesicle comprises abiodegradable lipid. In some embodiments, the multilamellar lipidvesicle comprises a phospholipid. In some embodiments, the multilamellarlipid vesicle comprises phosphocholine, phosphoglycerol, and/orphosphoethanolamine. In some embodiments, the multilamellar lipidvesicle comprises a functionalized lipid. In some embodiments, thefunctionalized lipid is a maleimide functionalized lipid. In someembodiments, the maleimide functionalized lipid is a maleimidefunctionalized phosphoethanolamine.

In some embodiments, the agent is a prophylactic agent. In someembodiments, the agent is a therapeutic agent. In some embodiments, theagent is an antigen. In some embodiments, the agent is an adjuvant. Insome embodiments, the vesicles comprise two or more agents. In someembodiments, the vesicles comprise an antigen and an adjuvant. In someembodiments, the vesicles comprise an antigen and two adjuvants. In someembodiments, the vesicles comprise a protein antigen and a TLR4 agonistand a TLR7 agonist. In some embodiments, the antigen such as the proteinantigen is present in the core of the vesicle and the adjuvant(s) arepresent in between the internal bilayers of the vesicle.

In some embodiments, the multilamellar lipid vesicle is conjugated toPEG on its external surface.

In some embodiments, the subject has or is at risk of developing cancer.In some embodiments, the subject has or is at risk of developing aninfection. In some embodiments, the subject has or is at risk ofdeveloping allergy or asthma or is experiencing or at risk ofexperiencing an asthmatic attack.

In some embodiments, the effective amount is an amount to stimulate animmune to response in the subject. The immune response may be a humoralresponse, or a cellular response, or it may be a combined humoral andcellular response. The cellular response may involve stimulation of CD8T cells.

In another aspect, the invention provides a method comprisingstimulating an immune response in a subject, in need thereof, byadministering multilamellar lipid vesicles having crosslinked lipidbilayers and comprising an antigen, wherein an effective amount of theantigen is administered to the subject. The multilamellar lipid vesicleshaving crosslinked lipid bilayers may be any of the foregoing suchvesicles or any of those described herein. The vesicles may furthercomprise one or more adjuvants. In some embodiments, the vesiclescomprise antigen, including protein antigen, in their cores andadjuvants in between their lipid bilayers. The protein antigen may be awhole protein antigen. The adjuvants may be but are not limited to TLR4agonists such as MPLA and TLR7 agonists such as R-848. In someembodiments, the vesicles comprise 300-400 μg of antigen per mg oflipid. In some embodiments, the vesicles are administered only once(i.e., a priming dose is sufficient). In some embodiments, the vesiclesare administered more than once (e.g., a prime and boost dose). In someembodiments, the antigen is a bacterial antigen, a viral antigen, afungal antigen, a parasitic antigen, or a mycobacterial antigen. In someembodiments, the antigen is a cancer antigen. In some embodiments, theimmune response is a synergistic immune response.

In another aspect, the invention provides a method comprising contactinga multilamellar lipid vesicle having crosslinked lipid bilayers and thatcomprises an agent, with a cell, or cell population, in vitro. The cellor cell population may be dendritic cells or other antigen presentingcells. The multilamellar lipid vesicle having crosslinked lipid bilayersmay be any of the foregoing multilamellar lipid vesicles havingcrosslinked lipid bilayers or any of those described herein.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A. Schematic of synthesis of interbilayer-crosslinkedmultilamellar vesicles (ICMVs). Divalent cation-mediated fusion leads toformation of multilamellar vesicles, in which adjacent bilayers werecrosslinked with DTT. The resulting ICMVs were PEGylated in a reactionwith PEG-thiol.

FIG. 1B. Illustration of ICMVs formed by conjugating two lipidheadgroups together in multilamellar vesicles.

FIGS. 2A-D. Synthesis of ICMVs. (A) Schematic illustration of ICMVsynthesis and cryoelectron microscope images: (i) Anionic,maleimide-functionalized liposomes are prepared from dried lipid films,(ii) divalent cations are added to induce fusion of liposomes and theformation of MLVs, (iii) membrane-permeable dithiols are added, whichcrosslink maleimide-lipids on apposed lipid bilayers in the vesiclewalls, and (iv) the resulting lipid particles are PEGylated withthiol-terminated PEG. Cryo-EM images from each step of the synthesisshow (i) initial liposomes, (ii) MLVs, and (iii) ICMVs with thick lipidwalls. Scale bars=100 nm. Right-hand image of (iii) shows a zoomed imageof an ICMV wall, where stacked bilayers are resolved as electron-densestriations; scale bar=20 nm. (B) ICMV particle size histogram measuredby dynamic light scattering. (C, D) Histograms of ICMV properties fromcryo-EM images show (C) the number of lipid bilayers per particle, and(D) the ratio of particle radius to lipid wall thickness. (n=165particles analyzed).

FIGS. 3A-B. (A) Histogram of particle diameters measured by dynamiclight scattering. (B) Cryo-EM image of ICMV particle.

FIGS. 4A-E. Protein encapsulation and release from ICMVs. (A)Encapsulation efficiency of the globular proteins SIV-gag, FLT-3L, orOVA in lipid vesicles collected at each step of ICMV synthesis. (B, C)Comparison of OVA encapsulation efficiency (B), and total proteinloading per particle mass (C) in ICMVs vs. dehydration-rehydrationvesicles (DRVs) or PLGA nanoparticles. (D) Kinetics of OVA release fromsimple liposomes, MLVs, or ICMVs (all with base lipid composition 4:5:1DOPC:MPB:DOPG) incubated in RPMI medium with 10% serum at 37° C.measured over 30 days in vitro. Also shown for comparison are releasekinetics for liposomes stabilized with cholesterol and PEG-lipid(38:57:5 DOPC:chol:PEG-DOPE). (E) Release of OVA from ICMVs was measuredin buffers simulating different aspects to of the endolysomalenvironment: reducing buffer, 100 mM beta-mercaptoethanol (beta-ME) inPBS; acidic buffer, 50 mM sodium citrate pH 5.0; lipase-containingbuffer, 500 ng/mL lipase A in PBS. Data represent the mean±s.e.m of atleast three experiments with n=3.

FIGS. 5A-B. Comparison of OVA encapsulation in traditional liposomes,lipid-coated PLGA particles, and ICMVs, showing (A) fraction of OVAencapsulated in particles and (B) the amount of OVA encapsulated pertotal particle mass.

FIGS. 6A-B 6. OVA released from sonicated liposomes, MLVs fused by Mg²⁺and ICMVs formed by Mg²⁺ and DTT in (A) PBS and (B) RPMI media with 10%serum.

FIGS. 7A-C. In vitro stimulation of immune responses by ICMVssupplemented with the TLR agonist MPLA. (A) Flow cytometry analysis ofexpression of the cell surface costimulatory markers CD40, CD80, andCD86 on splenic dendritic cells (DCs) after 18 hr incubation with 0.7μg/mL soluble OVA, equivalent doses of OVA loaded in ICMVs, or ICMVsloaded with an irrelevant protein (vivax malaria protein, VMP), in thepresence or absence of 0.1 μg/mL MPLA. (B) Splenic DCs were incubatedfor 18 hr with 10 μg/mL SIINFEKL peptide (OVA₂₅₇₋₂₆₄), 5.0 μg/mL solubleOVA, equivalent doses of OVA loaded in ICMVs, or VMP-loaded ICMVs in thepresence or absence of 0.05 μg/mL MPLA, and the extent ofcross-presentation of OVA was assessed by flow cytometry analysis ofcells stained with the 25-D1.16 mAb that recognizes SIINFEKL complexedwith H-2K^(b). (C) 5-(6)-carboxyfluorescein diacetate succinimidyldiester (CFSE)-labeled OVA-specific naïve OT-I CD8⁺ T-cells wereco-cultured with syngeneic splenic DCs pulsed with soluble 0.7 μg/mL OVAmixed with 0.1 μg/mL MPLA, or equivalent doses of OVA-loaded ICMVs mixedwith MPLA. Empty ICMVs without antigen or ICMVs loaded with theirrelevant antigen VMP were included as negative controls. Proliferationof CD8⁺ T-cells was assessed on day 3 by flow cytometry analysis of thedilution of CFSE in the OT-I CD8⁺ T-cells; shown are histograms of CFSEfluorescence. Gates on each histogram indicate the percentage of dividedcells in each sample. Data represent the mean±s.e.m of at least threeexperiments with n=3-4.

FIGS. 8A-B. (A) DC maturation and (B) activation in response toICMV-incorporated with MPLA and/or R-848.

FIGS. 9A-F. In vivo immunization with ICMVs vs. soluble antigen orantigen encapsulated in non-crosslinked vesicles. a, b, C57B1/6 micewere immunized subcutaneously (s.c.) with a single injection of 10 μgOVA delivered in soluble, liposomal, MLV, or ICMV formulations, eachmixed with 0.1 μg of MPLA. (A) The percentage of antigen-specific CD8⁺T-cells was determined by flow cytometry analysis of peripheral bloodmononuclear cells (PBMCs) 7 days post immunization with fluorescent OVApeptide-MHC tetramers. (B) Sera from the immunized mice were analyzed byELISA 21 days post immunization for OVA-specific IgG. (C, D) C57B1/6mice were injected with 10 μg of fluorophore-conjugated OVA mixed with0.1 μg of MPLA as a soluble, liposomal, or ICMV formulation, and thedraining inguinal lymph node (dLN) cells that internalized OVA wereassessed on day 2. (C) Shown are percentages of DCs (CD11c⁺),macrophages (F4/80⁺), B cells (B220⁺), and plasmacytoid DCs(CD11c⁺B220⁺) positive for OVA uptake, and (D) the mean fluorescenceintensity (MFI) of OVA⁺ populations. (E, F) C57B1/6 mice were injectedwith 10 μg of OVA mixed with 0.1 μg of MPLA as a soluble, liposomal, orICMV formulation, and 2 days later, DCs isolated from draining inguinalLNs were analyzed by flow cytometry to assess DC activation and antigencross-presentation. (E) Overlaid histograms show costimulatory markers(CD40 and CD86) and MHC-II expression in DCs. (F) The left panel showsoverlaid histograms of inguinal LN DCs stained for SIINFEKL-K^(b+)complexes, and mean MR levels are shown on the right panel. Datarepresent mean±s.e.m of 2-3 independent experiments conducted withn=3-4. *, p<0.05 and **, p<0.01, analyzed by one-way ANOVA, followed byTukey's HSD.

FIGS. 10A-B. (A) OVA-specific CD8+ T cells in peripheral blood after 7days of boost, as assessed by flow cytometry analysis of cells stainedwith antibodies against CD8 and peptide-MHC tetramers complexed with theOVA-derived peptide SIINFEKL. (B) Anti-OVA antibody titers measured onday 21 of immunization by ELISA.

FIG. 11A-E. ICMVs carrying antigen in the aqueous core and MPLA embeddedin the vesicle walls elicit potent antibody and CD8⁺ T-cell responses.(A) Schematic illustration of the vaccine groups: soluble OVA mixed withMPLA (MPLA), OVA-loaded ICMVs with MPLA only on the external surface(ext-MPLA ICMVs), or OVA-loaded ICMVs with MPLA throughout the lipidmultilayers (int-MPLA ICMVs). (B-G) C57B1/6 mice were immunized on days0, 21, and 35 at tail base s.c. with 10 μg OVA and either 0.1 μg or 1.0μg of MPLA formulated either as MPLA, ext-MPLA ICMVs, or int-MPLA ICMVs.(B) ELISA analysis of total OVA-specific IgG in sera. (C) Frequency ofOVA-specific T-cells in peripheral blood assessed over time via flowcytometry analysis of tetramer⁺CD8⁺ T-cells for vaccinations with 10 μgOVA and 0.1 μg MPLA. Response to vaccinations with soluble OVA+1 μg MPLAis (MPLA 10×) also shown for comparison. Shown are representative flowcytometry scatter plots from individual mice at day 41 and meantetramer⁺ values from groups of mice vs. time. (D) Analysis of T-celleffector/effector memory/central memory phenotypes in peripheral bloodby CD44/CD62L staining on tetramer⁺ cells from peripheral blood on day41. Shown are representative cytometry plots from individual mice andmean percentages of tet⁺CD44⁺CD62L⁺ cells among CD8⁺ T-cells at day 41.(E) Functionality of antigen-specific CD8⁺ T-cells was assayed on day 49with intracellular IFN-γ staining after ex vivo restimulation of PBMCswith OVA peptide in vitro. Representative flow cytometry histograms ofIFN-γ⁺CD8⁺ T-cells from individual mice and mean results from groups areshown. Data represent the mean±s.e.m of two independent experimentsconducted with n=3. c, *, p<0.05 compared to sol OVA+MPLA and #, p<0.05compared to ext-MPLA ICMVs. (D, E) *, p<0.05 and **, p<0.01, analyzed bytwo-way ANOVA, followed by Tukey's HSD.

FIGS. 12A-B. Alternative interbilayer-crosslinked reaction using “click”chemistry. (A) An alkyne-headgroup lipid was synthesized from DOPE andan alkyne precursor. (B) Liposomes were formed with alkyne-terminatedlipids and induced to form MLVs by Mg²⁺. Subsequent incubation of thealkyne-bearing MLVs with diazide and catalyst as indicated led tosuccessful formation of ICMVs with 83% particle yield, as measured afterparticle retrieval with low-speed centrifugation conditions.

DETAILED DESCRIPTION OF INVENTION

The invention provides stabilized multilamellar lipid vesicles for usein, inter alia, delivery of agents. Prior art vaccines based onrecombinant proteins avoid toxicity and anti-vector immunity associatedwith live vaccine (e.g., viral) vectors, but their immunogenicity ispoor, particularly for CD8⁺ T-cell (CD8T) responses. Synthetic particlescarrying antigens and adjuvant molecules have been developed to enhancesubunit vaccines, but in general these materials have failed to elicitCD8T responses comparable to live vectors in preclinical animal models.In contrast to these prior art compositions and methods, the inventionprovides stabilized multilamellar vesicles, such asinterbilayer-crosslinked multilamellar vesicles (ICMVs) formed bycrosslinking headgroups of adjacent lipid bilayers within multilamellarvesicles. These vesicles stably entrap, inter alia, protein antigens inthe vesicle core and lipid-based immunostimulatory molecules in thevesicle walls under extracellular conditions, but exhibited rapidrelease in the presence of endolysosomal lipases. When used to deliverantigen alone or in the presence of adjuvant, the vesicles of theinvention form an extremely potent vaccine (e.g., a whole-proteinvaccine), eliciting endogenous T-cell and antibody responses comparableto the strongest vaccine vectors.

The vesicles are stabilized by internal linking (e.g., crosslinking) oftheir lipid bilayers. The stabilized nature of these vesicles allowsthem to incorporate higher amounts of agents and to retain such agentsover a longer time period, as compared to simple liposomes or lipidcoated nano- or microparticles. Their sustained release kinetics,particularly in the presence of serum, make them useful in in vivodelivery of agents for which a slow, steady and prolonged release isdesirable or for which slow release in the extracellular environment butrapid release within cells is desirable. The invention contemplatesusing such vesicles with a number and variety of agents includingprophylactic agents, therapeutic agents, and/or diagnostic agents, asdescribed in greater detail herein. The invention therefore providescompositions comprising the afore-mentioned vesicles, methods for theirsynthesis, and methods for their use.

Stabilized Multilamellar Lipid Vesicles (MLV)

The invention provides MLV that are stabilized by linking adjacent (orapposed) lipid bilayers to one another. As used herein, a multilamellarvesicle is a nano- or microsphere having a shell that is comprised oftwo or more concentrically arranged lipid bilayers. As used herein,adjacent or apposed lipid bilayers (or lipid bilayer surfaces) intendbilayers or surfaces that are in close proximity to each other but thatare otherwise distinct and typically physically separate. This term doesnot typically mean the relationship between the two monolayers of asingle bilayer.

As used herein, “linking” means two entities stably bound to one anotherby any physiochemical means. Any linkage known to those of ordinaryskill in the art may be employed including covalent or noncovalentlinkage, although covalent linkage is preferred. In some importantembodiments described herein, covalent linkage between adjacent (orapposed) lipid bilayers in MLV is achieved through the use ofcrosslinkers and functionalized components of the lipid bilayer. Theinvention however contemplates that linking, including covalent linking,may be effected in other ways. As an example, the invention contemplatesmethods in which complementary reactive groups reside on components ofadjacent bilayer surfaces and linkage between the bilayer surfaces iseffected by reacting those groups to each other even in the absence of acrosslinker. Suitable complementary reactive groups are known in the artand described herein.

The interior of the vesicle is typically an aqueous environment, and itmay comprise an agent such as but not limited to a prophylactic,therapeutic or diagnostic agent. In some instances, the vesicles do notcomprise a solid core, such as a solid polymer core (e.g., a syntheticpolymer core). Instead, as discussed above, they may have a fluid corecomprising agents of interest. The core may comprise monomers forpolymerization into a hydrogel core in some instances. The vesicles mayalso be referred to herein as particles, including nano- ormicroparticles, although it is to be understood that such nano- ormicro-particles have the attributes of the stabilized MLVs andinterbilayer crosslinked multilamellar lipid vesicles (ICMVs) of theinvention.

The vesicles may have a void volume at their core and/or they maycomprise one or more agents in their core and/or between adjacent (orapposing) lipid bilayers, as shown in FIGS. 1A, 1B and 2A. The agentsare typically included in the lipid solution during the synthesisprocess and in this manner are incorporated (e.g., by encapsulation)into the vesicles during synthesis. Lipophilic molecules may also beincorporated directly into the lipid bilayers as the vesicles are formedor molecules with lipophilic tails may be anchored to the lipid bilayersduring vesicle formation. The vesicles may be produced in the absence ofharsh solvents, such as organic solvents, and as a result they may beable to encapsulate a wide variety of agents including those that wouldbe susceptible to organic solvents and the like.

The amount of agent in the vesicles may vary and may depend on thenature of the agent. As demonstrated in the Examples, 300-400 μg ofprotein agent per mg of lipid may be incorporated into the vesicles ofthe invention. In some embodiments, the vesicles may comprise about 100μg of agent, or about 150 μg of agent, or about 200 μg of agent, orabout 250 μg of agent, or about 300 μg of agent, or about 325 μg ofagent, or about 350 μg of agent, or about 375 μg of agent, or about 400μg of agent, or about 410 μg of agent, per mg of lipid. In someembodiments, the agent may be a protein such as a protein antigen.

The vesicles of the invention may also be characterized by theirretention profiles. In some embodiments, the vesicles release agent at arate of about 25% per week when placed in serum containing media (e.g.,10% serum) and maintained at 37° C. In some embodiments, the vesiclesrelease about 25% of agent in the first week and up to about 90% afterabout 30 days under these conditions. In some embodiments, the vesiclesmaintain at least 80%, at least 85%, at least 90%, or at least 95% oftheir agent when stored in buffer (such as PBS) at 4° C. for 30 days.

The number of lipid bilayers in each vesicle may vary, with a typicalrange of at least 2 to about 50, or at least 2 to about 25, or at least2 to about 15, or at least 2 to about 10, or at least 2 to about 5.

The diameter of the vesicles may vary. In some instances, the vesicleswill have a diameter ranging from about 100 to about 500 nm, includingfrom about 125 to about 300 nm, including from about 150 to about 300nm, including from about 175 to about 275 nm. In some instances, thediameter ranges from about 150 to about 250 nm. The diameter profilesfor ICMV prepared as described in the Examples is shown in FIGS. 2B and3A. It will be understood that, in any preparation of vesicles, therewill be heterogeneity between vesicles relating to vesicle diameter,number of lipid bilayers, amount of loaded agent, etc. Suchdistributions are shown in the Examples.

As used herein, the vesicles of the invention may also be referred to asliposomes (e.g., stabilized multilamellar liposomes or, as discussedbelow, interbilayer crosslinked multilamellar liposomes). Accordingly,the use of the term “vesicles” is not intended to convey source ororigin of the vesicles. The vesicles of the invention are syntheticvesicles (i.e., they are produced in vitro), as will be discussed ingreater detail below.

The vesicles may be isolated, intending that they are physicallyseparated in whole or in part from the environment in which they aresynthesized. As an example, vesicles comprising an agent (i.e., their“cargo” or “payload”) may be separated in whole or in part from vesicleslacking agent (i.e., empty vesicles), and may then be referred to as“isolated vesicles.” Separation may occur based on weight (or mass),density (including buoyant density), size, color and the like (e.g.,where the cargo of the vesicle is detectable by its energy emission),etc. As described in the Examples, centrifugation can be used toseparate vesicles of the invention from simple liposomes or MLVs ofidentical lipid composition that do not have crosslinked bilayers.Centrifugation at about 14,000 g for about 4 minutes is sufficient toseparate the vesicles of the invention, which pellet, from these otherparticle types.

Interbilayer Crosslinked Multilamellar Lipid Vesicles

An example of the stabilized MLV of the invention is the interbilayercrosslinked multilamellar (lipid) vesicles (ICMV). Like the stabilizedMLV described above, ICMV are nano- or microspheres having a shell thatis comprised of two or more concentrically arranged lipid bilayers thatare conjugated to each other as described herein. The number of lipidbilayers to in the stabilized multilamellar vesicles, including theICMV, may vary from about 2-30, but is more commonly in the range of2-15. Accordingly, in various embodiments, the number of layers may be2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. The frequencydistribution of bilayer numbers resulting from one exemplary synthesisof ICMV is shown in FIG. 2C. The bilayers are typically comprised oflipids having hydrophilic heads and hydrophobic tails that are arrangedin a manner similar to a cell membrane (i.e., with the hydrophilic headsexposed to typically an aqueous environment and the hydrophobic tailsburied in the bilayer).

The ICMV are stabilized via crosslinks between their lipid bilayers, andthey are therefore referred to as “interbilayer crosslinked” MLV. Asused herein, this means that at least two lipid bilayers in the shell ofthe vesicle are crosslinked to each other. The crosslinked bilayers aretypically those that are apposed or adjacent to each other. Most or allof the lipid bilayers in the shell may be crosslinked to their apposinglipid bilayer in the shell. There may be one or more crosslinks betweenlipid bilayers. Typically, there will be numerous crosslinks betweenlipid bilayers. The arrangement and positioning of such crosslinks maybe random or non-random. The degree of crosslinks (and thus theresultant stability of the vesicles) will depend upon the proportion offunctionalized lipids (or other lipid bilayer components) used to makethe vesicles and the crosslinking conditions (including, for example,time of incubation of the vesicles with a crosslinker). It will beunderstood that the higher the proportion of functionalized lipids (orother lipid bilayer components) in the vesicles, the more crosslinksthat will be formed, all other factors and parameters being equal.Similarly, the more favorable the conditions towards crosslinking, thegreater degree of crosslinking that will be achieved.

Synthesis Methods

An exemplary synthesis method is as follows: Lipids and optionally otherbilayer components are combined to form a homogenous mixture. This mayoccur through a drying step in which the lipids are dried to form alipid film. The lipids are then combined (e.g., rehydrated) with anaqueous solvent. The aqueous solvent may have a pH in the range of about6 to about 8, including a pH of about 7. Buffers compatible with vesiclefusion are used, typically with low concentrations of salt. The solventused in the Examples is a 10 mM bis-tris propane (BTP) buffer pH 7.0.The nature of the buffer may impact the length of the incubation, asshown in Table 1A. For example, a buffer such as PBS may require alonger incubation time as compared to a buffer such as BTP, all otherthings being equal. If the buffer is PBS, then the incubation times maybe about 6-24 hours, or 8-16 hours, or 10-12 hours. If the buffer isBTP, then the incubation times may be shorter including 1-4 hours, or1-2 hours. Accordingly a variety of aqueous buffers may be used providedthat a sufficient incubation time is also used. This step may alsoinclude the presence of the agent(s) to be incorporated into thevesicles. The resultant liposomes are then incubated with one or moredivalent cations in order to fuse them into multilamellar vesicles.Suitable divalent cations include Mg²⁺, Ca²⁺, Ba²⁺, or Sr²⁺. Multivalentor polymeric cations could also be employed for vesicle fusion. Vesiclefusion could also be achieved via the mixing of cationic vesicles withdivalent or higher valency anions; an example would be fusion ofcationic liposomes with DNA oligonucleotides or DNA plasmids. This maybe done under agitation such as sonication, vortexing, and the like. Ifthe liposomes were made in the presence of an agent, the MLVs willcomprise the agent in their core and/or between the concentricallyarranged lipid bilayers. The invention contemplates fusion of liposomescarrying different agents to form MLVs that comprise such agents.

The resultant MLVs are then incubated with a crosslinker, and preferablya membrane-permeable crosslinker. As stated herein, the nature of thecrosslinker will vary depending on the nature of the reactive groupsbeing linked together. As demonstrated in the Examples, adithiol-containing crosslinker such as DTT or(1,4-Di-[3′-(2′-pyridyldithio)-propionamido]butane) may be used tocrosslink MLVs comprised of maleimide functionalized lipids (or otherfunctionalized lipid bilayer components), or diazide crosslinkers couldbe used to crosslink alkyne headgroup lipids via “click” chemistry, asshown in FIG. 12. These various incubations are all carried out underaqueous conditions at a pH in the range of about 6 to about 8, or about6.5 to about 7.5, or at about 7. The crosslinking step may be performedat room temperature (e.g., 20-25° C.) or at an elevated temperatureincluding for example up to or higher than 37° C.

The resultant crosslinked vesicles may then be collected (e.g., bycentrifugation or other pelleting means), washed and then PEGylated ontheir outermost or external surface (e.g., as used herein, the vesiclesmay be referred to “surface-PEGylated” or “surface-conjugated” to PEG)by incubation with a thiol-PEG. The PEG may be of any size, includingbut not limited to 0.1-10 kDa, 0.5-5 kDa, or 1-3 kDa. A 2 kDa PEGfunctionalized with thiol is used in the Examples. The incubation periodmay range from about 10 minutes to 2 hours, although it may be shorteror longer depending on other conditions such as temperature,concentration and the to like. The PEGylation step may be performed atroom temperature (e.g., 20-25° C.) or at an elevated temperatureincluding for example up to or higher than 37° C. A 30 minute incubationperiod is used in the exemplary synthesis methods of the Examples. Thevesicles then may be collected (e.g., by centrifugation or otherpelleting means) and washed with water or other aqueous buffer.

The vesicles may be stored at 4° C. in a buffered solution such as butnot limited to PBS or they may be lyophilized in the presence ofsuitable cryopreservants and then stored at −20° C. Suitablecryopreservants include those that include sucrose (e.g., a 1-5%sucrose, and preferably about 3% sucrose solution).

Crosslinking could also be achieved by coupling between a reactive groupin one bilayer with a complementary reactive group in the adjacentbilayer. For example, fused vesicles containing succinimidylester-functionalized lipid (A) headgroups and primary-amine-containing(B) headgroups could achieve crosslinking by in situ reaction betweenthe A and B lipids of adjacent bilayers. A variety of othercomplementary functionalized lipids familiar to those skilled in the artcould be employed in a similar manner.

The molar ratio of functionalized lipid (or other functionalizedcomponent of the lipid bilayer) to crosslinker may vary depending on theconditions. In some instances, it may range from about 1 to about 5. Insome embodiments, a molar ratio of 2 is sufficient (i.e., the molarratio of functionalized lipid (or component) to crosslinker is 2:1). TheExamples describe a synthesis method in which a 2:1 molar ratio ofmaleimide functionalized lipid to DTT is used to crosslink the lipidbilayers of the vesicles. The incubation time may range from 1 hour to24 hours, from 2-18 hours, from 2 to 12 hours, or from 2 to 6 hours. Insome instances, it may be about 2 hours. In other instances, it may beovernight (e.g., about 12 hours).

The molar % of the functionalized lipid in the vesicles may range from1% to 100%, or from about 10% to about 60% in some instances, or fromabout 25% to about 55% in some instances. In some instances, the molar %of the functionalized lipid in the vesicles is typically at least 10%,preferably at least 15%, more preferably at least 20%, and even morepreferably at least 25%. Tables 1A and 1B show that in the absence offunctionalized lipid, no vesicles are formed.

Conversely, the non-functionalized lipids may be present at about 0% to99% as a molar %. More typically, the non-functionalized lipids may bepresent at about 40%-75% or 40% to 60% as a molar %.

In one important embodiment, the vesicles are synthesized using DOPC,DOPG and maleimide-functionalized DSPE. The ratio of these lipids toeach other may vary. Tables 1A and 1B show the effects of varying theratio of DOPC:DOPG:maleimide functionalized lipid on vesicle yield. Themolar % of DOPC may range from 1-50%, the molar % of DOPG may range from1-50%, and the molar % of the maleimide functionalized lipid may rangefrom 1-80%. Some embodiments of the invention provide vesicles having aDOPC:DOPG:maleimide functionalized lipid ratio of 40:10:50. Someembodiments provide vesicles having a DOPC:DOPG:maleimide functionalizedlipid ratio of 60:15:25. Some embodiments provide vesicles comprised ofDOPG and a maleimide functionalized lipid.

Lipids

The vesicles are comprised of one or more lipids. The type, number andratio of lipids may vary with the proviso that collectively they formspherical bilayers (i.e., vesicles). The lipids may be isolated from anaturally occurring source or they may be synthesized apart from anynaturally occurring source.

At least one (or some) of the lipids is/are amphipathic lipids, definedas having a hydrophilic and a hydrophobic portion (typically ahydrophilic head and a hydrophobic tail). The hydrophobic portiontypically orients into a hydrophobic phase (e.g., within the bilayer),while the hydrophilic portion typically orients toward the aqueous phase(e.g., outside the bilayer, and possibly between adjacent apposedbilayer surfaces). The hydrophilic portion may comprise polar or chargedgroups such as carbohydrates, phosphate, carboxylic, sulfato, amino,sulfhydryl, nitro, hydroxy and other like groups. The hydrophobicportion may comprise apolar groups that include without limitation longchain saturated and unsaturated aliphatic hydrocarbon groups and groupssubstituted by one or more aromatic, cyclo-aliphatic or heterocyclicgroup(s). Examples of amphipathic compounds include, but are not limitedto, phospholipids, aminolipids and sphingolipids.

Typically, the lipids are phospholipids. Phospholipids include withoutlimitation phosphatidylcholine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and thelike. It is to be understood that other lipid membrane components, suchas cholesterol, sphingomyelin, cardiolipin, etc. may be used.

The lipids may be anionic and neutral (including zwitterionic and polar)lipids including anionic and neutral phospholipids. Neutral lipids existin an uncharged or neutral zwitterionic to form at a selected pH. Atphysiological pH, such lipids include, for example,dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,cholesterol, cerebrosides and diacylglycerols. Examples of zwitterioniclipids include without limitation dioleoylphosphatidylcholine (DOPC),dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine(DOPS). An anionic lipid is a lipid that is negatively charged atphysiological pH. These lipids include without limitationphosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein asnon-cationic lipids. Such lipids may contain phosphorus but they are notso limited. Examples of non-cationic lipids include lecithin,lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine,dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE),palmitoyloleoyl-phosphatidylethanolamine (POPE)palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), phosphatidylserine,phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, and cholesterol.

Additional nonphosphorous containing lipids include stearylamine,dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate,hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers,triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylatedfatty acid amides, dioctadecyldimethyl ammonium bromide and the like,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, and cerebrosides. Lipids such aslysophosphatidylcholine and lysophosphatidylethanolamine may be used insome instances. Noncationic lipids also include polyethyleneglycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycolconjugated to phospholipids or to ceramides (referred to as PEG-Cer).

In some instances, modified forms of lipids may be used including formsmodified with detectable labels such as fluorophores. In some instances,the lipid is a lipid analog that emits signal (e.g., a fluorescentsignal). Examples include without limitation1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD).

Preferably, the lipids are biodegradable in order to allow release ofencapsulated agent in vivo and/or in vitro. Biodegradable lipids includebut are not limited to 1,2-dioleoyl-sn-glycero-3-phosphocholine(dioleoyl-phosphocholine, DOPC), anionic1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1′-rac-glycerol)(dioleoyl-phosphoglycerol, DOPG), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine(distearoyl-phosphoethanolamine, DSPE). Non-lipid membrane componentssuch as cholesterol may also be incorporated.

Functionalized Lipids or Bilayer Components

At least one component of the lipid bilayer must be functionalized (orreactive). As used herein, a functionalized component is a componentthat comprises a reactive group that can be used to crosslink adjacentbilayers of the multilamellar vesicle. The bilayer component may bemodified to comprise the reactive group.

One or more of the lipids used in the synthesis of the vesicles may befunctionalized lipids. As used herein, a functionalized lipid is a lipidhaving a reactive group that can be used to crosslink adjacent bilayersof the multilamellar vesicle. In some embodiments, the reactive group isone that will react with a crosslinker (or other moiety) to formcrosslinks between such functionalized lipids (and thus between lipidbilayers in the vesicle). The reactive group may be located anywhere onthe lipid that allows it to contact a crosslinker and be crosslinked toanother lipid in an adjacent apposed bilayer. In some embodiments, it isin the head group of the lipid, including for example a phospholipid. Anexample of a reactive group is a maleimide group. Maleimide groups maybe crosslinked to each other in the presence of dithiol crosslinkerssuch as but not limited to dithiolthrietol (DTT). An example of afunctionalized lipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide, referred to herein as MPB. The Examples demonstrate use ofthis functionalized lipid in the synthesis of vesicles of the invention.Another example of a functionalized lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000] (also referred to as maleimide-PEG 2k-PE). Another exampleof a functionalized lipid is dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal).

It is to be understood that the invention contemplates the use of otherfunctionalized lipids, other functionalized lipid bilayer components,other reactive groups, and other crosslinkers. In addition to themaleimide groups, other examples of reactive groups include but are notlimited to other thiol reactive groups, amino groups such as primary andsecondary amines, carboxyl groups, hydroxyl groups, aldehyde groups,alkyne groups, azide groups, carbonyls, haloacetyl (e.g., iodoacetyl)groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydrylgroups, pyridyl disulfide groups, and the like.

Functionalized and non-functionalized lipids are available from a numberof commercial sources including Avanti Polar Lipids (Alabaster, Ala.).

It is to be understood that the invention contemplates various ways tolink adjacent bilayers in the multilamellar vesicles to each other. Insome instances, crosslinkers are used to effect linkage between adjacentbilayers. The invention however is not so limited.

As an example, vesicles may be formed using click chemistry. Anexemplary synthesis method uses alkyne-modified lipids and alkyne-azidechemistry, as follows. Alkyne-modified lipids were made by mixing thelipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 744mg, 1 mmol) with N-hydroxysuccinimide ester of propiolic acid (167 mg, 1mmol) and Et₃N (202 mg, 2 mmol) in 5 mL CDCl₃. The reaction wasmonitored by NMR. After 3 hours at room temperature, the reaction wascompleted. After the organic solution was washed with 5 mL 5% Na₂CO₃, 1%HCl and brine, dried under Na₂SO₄ and evaporated, and alkyne-modifiedDOPE was weighed. 1.26 μmol of lipid film with DOPC and alkyne-DOPE in1:1 molar ratio was prepared, hydrated, sonicated, and induced to fusewith 10 mM Mg²⁺ as described previously. MLVs with alkyne-functionalizedlipids were incubated with 2.5 mM CuSO₄, copper wire, and 1.5 mM 1,14-diazido-3,6,9,12-tetraoxatetradecane for 24 hours at roomtemperature. Particle yield was measured after 3× washes withcentrifugation.

Crosslinkers

The crosslinker may be a homobifunctional crosslinker or aheterobifunctional crosslinker, depending upon the nature of reactivegroups in the lipid bilayers that are being linked to each other. Theterms “crosslinker” and “crosslinking agent” are used interchangeably toherein. Homobifunctional crosslinkers have two identical reactivegroups. Heterobifunctional crosslinkers have two different reactivegroups.

In one instance, adjacent bilayers are crosslinked to each other usingthe same functionalized lipid (or other bilayer component) and acrosslinker (such as a homobifunctional crosslinker). In anotherinstance, adjacent bilayers are crosslinked to each other usingdifferent functionalized lipids (or other bilayer components) and acrosslinker (such as a heterobifunctional crosslinker).

Various types of commercially available crosslinkers are reactive withone or more of the following groups: maleimides, primary amines,secondary amines, sulphydryls, carboxyls, carbonyls and carbohydrates.Examples of amine-specific crosslinkers are bis(sulfosuccinimidyl)suberate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidylsuberate, disuccinimidyl tartarate, dimethyl adipimate.2 HCl, dimethylpimelimidate.2 HCl, dimethyl suberimidate.2 HCl, and ethyleneglycolbis-[succinimidy-[succinate]]. Crosslinkers reactive withsulfhydryl groups include bismaleimidohexane,1,4-di-[3′-(2′-pyridyldithio)-propionamido)]butane,1-[p-azidosalicylamido]-4-[iodoacetamido]butane, andN-[4-(p-azidosalicylamido)butyl]-3′-[2′-pyridyldithio]propionamide.Crosslinkers preferentially reactive with carbohydrates includeazidobenzoyl hydrazine. Crosslinkers preferentially reactive withcarboxyl groups include 4[p-azidosalicylamido]butylamine. Dithiolcrosslinkers such as dithiolthietol (DTT),1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane (DPDPB), and in someinstances thiol containing polymers such as (PEG)-SH2 can be used tocrosslink maleimide reactive groups. The structure of DTT is

The structure of DPDPB is

Crosslinkers reactive with alkyne groups include diazides, such as 1,14-Diazido-3,6,9,12-Tetraoxatetradecane, and other groups compatiblewith “click” chemistry.

Heterobifunctional crosslinkers that react with amines and sulfhydrylsinclude N-succinimidyl-3-[2-pyridyldithio]propionate,succinimidyl[4-iodoacetyl]aminobenzoate, succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl to6-[3-[2-pyridyldithio]propionamido]hexanoate, and sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Heterobifunctionalcross-linkers that react with carboxyl and amine groups include1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.Heterobifunctional crosslinkers that react with carbohydrates andsulfhydryls include4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide.2 HCl,4-(4-N-maleimidophenyl)-butyric acid hydrazide.2 HCl, and3-[2-pyridyldithio]propionyl hydrazide. Other crosslinkers arebis-[β-4-azidosalicylamido)ethyl]disulfide and glutaraldehyde.

Crosslinkers are also preferably membrane permeable (or lipid soluble)so that they may diffuse through one or more bilayers of the MLVs toeffect cros slinking between various adjacent layers. Any weaklypolar/uncharged bifunctional or heterobifunctional small molecule may bean effective membrane permeable crosslinker, particularly if suchmolecule comprises a reactive group such as but not limited tomaleimides, succinimidyl esters, azides, thiols, and the like. Examplesof membrane permeable crosslinkers include but are not limited to DTTand 1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane (DPDPB).

PEGylation

The ICMVs may be further modified. As described in the Examples, theICMV may be conjugated to polyethylene glycol (PEG) on their surface.PEGylation is used clinically to increase the half-life of variousagents including STEALTH liposomes. PEGylation may be accomplished byreacting functionalized lipids on the surface of the stabilized MLVswith a complementary functionalized PEG. The lipids are preferably notconjugated to PEG prior to ICMV synthesis, and rather PEG is conjugatedto the ICMV external surface post-synthesis or PEG-lipid conjugates areintroduced into the external membrane layer of the particles by“post-insertion” processes.

Reactive groups to be used to PEGylate the ICMVs may be the same asthose used to crosslink the bilayers, in which case no additionalfunctionalized lipids (or other functionalized components) are required.As an example, if the ICMVs comprise maleimide functionalized lipids,then the functionalized PEG may be thiol-PEG. Alternatively, thereactive groups used to stabilize the vesicles may be different fromthose used to conjugate PEG to the external surface. Those of ordinaryskill in the art will appreciate that other modified versions of PEG maybe used depending on the nature of the reactive group in thefunctionalized lipid (or component) in the lipid bilayer of thevesicles. Suitable reactive groups include without limitation aminogroups such as primary and secondary amines, carboxyl groups, sulfhydrylgroups, hydroxyl groups, aldehyde groups, azide groups, carbonyls,maleimide groups, haloacetyl (e.g., iodoacetyl) groups, imidoestergroups, N-hydroxysuccinimide esters, and pyridyl disulfide groups.

Table 2 illustrates the effect of PEGylation on the ICMV diameterimmediately after synthesis and after 7 days in PBS Immediately aftersynthesis, the PEGylated ICMVs have a slightly larger diameter thannon-PEGylated ICMVs (e.g., 272+/−39 nm for the PEGylated ICMVs versus234+/−45 nm for the non-PEGylated in one experiment).

Various tests may be performed on the resultant stabilized vesicles inorder to determine, inter alia, size and surface charge (e.g., bydynamic light scattering), fraction of lipids on their external surface(e.g., by lamellarity assay), amount of agent incorporated therein ortherethrough (e.g., by FACS), and the like. Other tests that may beperformed on the vesicles include confocal microscopy and cryo-tunnelingelectron microscopy (TEM).

The following Tables provide the results of various tests performed onthe stabilized vesicles.

TABLE 1A ICMV yield with varying synthesis conditions. Yield Lipid Incu-% composition Buffer Cation DTT bation 40 DOPC/G/MPB 10 mM BTP 10 mMMgCl2 1.5 mM 2 hr (40:10:50) pH 7.0 45 DOPC/G/MPB 10 mM BTP 10 mM CaCl21.5 mM 2 hr (40:10:50) pH 7.0 0 DOPC/G/MPB 10 mM BTP 10 mM MgCl2 2 hr(40:10:50) pH 7.0 5 DOPC/G/MPB 10 mM BTP  15 mM 2 hr (40:10:50) pH 7.0 5DOPC/G/MPB 10 mM BTP 1.5 mM 2 hr (40:10:50) pH 7.0 0 DOPC/G/MPB 10 mMBTP 20 mM NaCl 1.5 mM 2 hr (40:10:50) pH 7.0 0 DOPC/G/MPB PBS 10 mMMgCl2 1.5 mM 2 hr (40:10:50) pH 7.0 40 DOPC/G/MPB PBS 10 mM MgCl2 1.5 mMO/N (40:10:50) pH 7.0 45 DOPC/G/MPB 10 mM BTP 10 mM MgCl2 1.5 mM 2 hr(40:10:50) pH 7.0 15 DOPC/G/MPB 10 mM BTP 10 mM MgCl2 1.5 mM 2 hr(60:15:25) pH 7.0 0 DOPC/G/MPB 10 mM BTP 10 mM MgCl2 1.5 mM 2 hr(72:18:10) pH 7.0 0 DOPC/DOPG 10 mM BTP 10 mM MgCl2 1.5 mM 2 hr (80:20)pH 7.0 0 DOPC 10 mM BTP 10 mM MgCl2 1.5 mM 2 hr pH 7.0

TABLE 1B ICMV yield with varying synthesis conditions. Lipid composition(molar ratio)^(a) Cation^(b) Crosslinker^(c) Yield ^(d)

1 DOPC/DOPG/MPB MgCl₂ DTT 45 (40:10:50) 2 DOPC/DOPG/MPB CaCl₂ DTT 50(40:10:50) 3 DOPC/DOPG/MPB 20 mM NaCl DTT 0 (40:10:50) 4 DOPC/DOPG/MPBMgCl₂ — 0 (40:10:50) 5 DOPC/DOPG/MPB — DTT 7 (40:10:50) 6 DOPC/DOPG/MPB— 15 mM DTT 4 (40:10:50) 7 DOPC/DOPG/MPB MgCl₂ DTT 15 (60:15:25) 8DOPC/DOPG/MPB MgCl₂ DTT 0 (72:18:10) 9 DOPC/DOPG MgCl₂ DTT 0 (80:20) 10DOPC/DOPG/MPB MgCl₂ DPDPB^(e) 48 (40:10:50) 11 DOPC/DOPG/MPB MgCl₂(PEG)-SH₂ ^(f) 3 (40:10:50) ^(a)hydrated with 10 mM bis-tris propane atpH 7.0; ^(b)at 10 mM unless noted otherwise; ^(c)at 1.5 mM unless notedotherwise ^(d) percentage of lipid mass recovered after synthesis andcentrifugation at 14,000 x g for 4 min^(e)1,4-Di-[3′-(2′pyridyldithio)-propionamido]butane (MW 482); ^(f)MW2000

indicates data missing or illegible when filed

TABLE 2 Vesicle characterization at each step in synthesis process.Diameter Diameter after Synthesis Hydrodynamic Zeta after 7 daysDiameter after lyophilization step diameter^(a) Polydispersity potentialin 4_(?)C lyophilization with 3% sucrose (FIG. 2A) Samples (nm) index(mV) (nm) (nm) (nm) (i) Liposomes 192 ± 39 0.385 ± 0.11  −0.141 ± 0.44N/A N/A N/A (ii) Mg²⁺-fused 220 ± 26 0.217 ± 0.053 −0.151 ± 0.67 N/A N/AN/A MLVs (iii) ICMVs 244 ± 17 0.223 ± 0.11  −0.415 ± 0.33 1610 ± 570 N/AN/A (iv) PEGylated 263 ± 20 0.183 ± 0.025  −2.34 ± 0.44 265 ± 27 2960 ±1800 269 ± 41 ICMVs ^(a)measured by dynamic light scattering (DLS)^(b)Fraction of lipid exposed on the external surface of vesiclesdecreased after interbilayer-crosslinked as measured by lamellarityassay (see Lutsiak et al. Pharm Res 19, 1480-1487 (2002)) *all valueswith mean ± SD

Agents

The invention contemplates the delivery, including in some instancessustained delivery, of agents to regions, tissues or cells in vivo or invitro using the stabilized lipid vesicles, including the ICMV, of theinvention. As used herein, an agent is any atom or molecule or compoundthat can be used to provide benefit to a subject (including withoutlimitation prophylactic or therapeutic benefit) or that can be used fordiagnosis and/or detection (for example, imaging) in vivo or that hasuse in in vitro applications.

Any agent may be delivered using the compositions (e.g., the stabilizedMLVs such as the ICMVs, and compositions thereof includingpharmaceutical compositions thereof) and methods of the inventionprovided that it can be encapsulated into (including throughout) orotherwise carried on the stabilized MLVs such as the ICMVs providedherein. For example, the agent must be able to withstand the synthesisand optionally storage process for these vesicles. The vesicles may besynthesized and stored in, for example, a lyophilized form, preferablywith a sucrose based excipient. The agents, if incorporated into thevesicles during synthesis, should be stable during such storageprocedures and times.

The agent may be without limitation a protein, a polypeptide, a peptide,a nucleic acid, a small molecule (e.g., chemical, whether organic orinorganic) drug, a virus-like particle, a steroid, a proteoglycan, alipid, a carbohydrate, and analogs, derivatives, mixtures, fusions,combinations or conjugates thereof. The agent may be a prodrug that ismetabolized and thus converted in vivo to its active (and/or stable)form.

The agents may be naturally occurring or non-naturally occurring.Naturally occurring agents include those capable of being synthesized bythe subjects to whom the vesicles are administered. Non-naturallyoccurring are those that do not exist in nature normally, whetherproduced by plant, animal, microbe or other living organism.

One class of agents is peptide-based agents such as (single ormulti-chain) proteins and peptides. Examples include antibodies, singlechain antibodies, antibody fragments, enzymes, co-factors, receptors,ligands, transcription factors and other regulatory factors, someantigens (as discussed below), cytokines, chemokines, and the like.These peptide-based agents may or may not be naturally occurring butthey are capable of being synthesized within the subject, for example,through the use of genetically engineered cells.

Another class of agents that can be delivered using the vesicles of theinvention includes those agents that are not peptide-based. Examplesinclude chemical compounds that are non-naturally occurring, or chemicalcompounds that are not naturally synthesized by mammalian (and inparticular human) cells.

A variety of agents that are currently used for therapeutic ordiagnostic purposes can be delivered according to the invention andthese include without limitation imaging agents, immunomodulatory agentssuch as immunostimulatory agents and immunoinhibitory agents, antigens,adjuvants, cytokines, chemokines, anti-cancer agents, anti-infectiveagents, nucleic acids, antibodies or fragments thereof, fusion proteinssuch as cytokine-antibody fusion proteins, to Fc-fusion proteins, andthe like.

Imaging Agents.

As used herein, an imaging agent is an agent that emits signal directlyor indirectly thereby allowing its detection in vivo. Imaging agentssuch as contrast agents and radioactive agents that can be detectedusing medical imaging techniques such as nuclear medicine scans andmagnetic resonance imaging (MRI). Imaging agents for magnetic resonanceimaging (MRI) include Gd(DOTA), iron oxide or gold nanoparticles;imaging agents for nuclear medicine include ²⁰¹Tl, gamma-emittingradionuclide 99 mTc; imaging agents for positron-emission tomography(PET) include positron-emitting isotopes, (18)F-fluorodeoxyglucose((18)FDG), (18)F-fluoride, copper-64, gadoamide, and radioisotopes ofPb(II) such as 203 Pb, and 11In; imaging agents for in vivo fluorescenceimaging such as fluorescent dyes or dye-conjugated nanoparticles. Inother embodiments, the agent to be delivered is conjugated, or fused to,or mixed or combined with an imaging agent.

Immunostimulatory Agents.

As used herein, an immunostimulatory agent is an agent that stimulatesan immune response (including enhancing a pre-existing immune response)in a subject to whom it is administered, whether alone or in combinationwith another agent. Examples include antigens, adjuvants (e.g., TLRligands such as imiquimod and resiquimod, imidazoquinolines, nucleicacids comprising an unmethylated CpG dinucleotide, monophosphoryl lipidA (MPLA) or other lipopolysaccharide derivatives, single-stranded ordouble-stranded RNA, flagellin, muramyl dipeptide), cytokines includinginterleukins (e.g., IL-2, IL-7, IL-15 (or superagonist/mutant forms ofthese cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand,etc.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28,anti-CD3, or single chain/antibody fragments of these molecules), andthe like.

Antigens.

The antigen may be without limitation a cancer antigen, a self orautoimmune antigen, a microbial antigen, an allergen, or anenvironmental antigen. The antigen may be peptide, lipid, orcarbohydrate in nature, but it is not so limited.

Cancer Antigens.

A cancer antigen is an antigen that is expressed preferentially bycancer cells (i.e., it is expressed at higher levels in cancer cellsthan on non-cancer cells) and in some instances it is expressed solelyby cancer cells. The cancer antigen may be expressed within a cancercell or on the surface of the cancer cell. The cancer antigen may beMART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP,cyclophilin b, colorectal associated antigen (CRC)—C017-1A/GA733,carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostatespecific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membraneantigen (PSMA), T cell receptor/CD3-zeta chain, and CD20. The cancerantigen may be selected from the group consisting of MAGE-AL MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-All, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05). The cancerantigen may be selected from the group consisting of GAGE-1, GAGE-2,GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9. The cancerantigen may be selected from the group consisting of BAGE, RAGE, LAGE-1,NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras,RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin,p120ctn, gp100^(Pmel117), PRAME, NY-ESO-1, cdc27, adenomatous polyposiscoli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2ganglioside, GD2 ganglioside, human papilloma virus proteins, Smadfamily of tumor antigens, lmp-1, PIA, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, and c-erbB-2.

Microbial Antigens.

Microbial antigens are antigens derived from microbial species such aswithout limitation bacterial, viral, fungal, parasitic and mycobacterialspecies. As such, microbial antigens include bacterial antigens, viralantigens, fungal antigens, parasitic antigens, and mycobacterialantigens. Examples of bacterial, viral, fungal, parasitic andmycobacterial species are provided herein. The microbial antigen may bepart of a microbial species or it may be the entire microbe.

Allergens.

An allergen is an agent that can induce an allergic or asthmaticresponse in a subject. Allergens include without limitation pollens,insect venoms, animal dander dust, fungal spores and drugs (e.g.penicillin). Examples of natural, animal and plant allergens include butare not limited to proteins specific to the following genera: Canine(Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae);Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g.Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alternata); Alder; Alnus (Alnusgultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea(Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantagolanceolata); Parietaria (e.g. Parietaria officinalis or Parietariajudaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apismultiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparisobtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g.Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticumaestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festucaelatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avenasativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthumodoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g.Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

Adjuvants.

The adjuvant may be without limitation alum (e.g., aluminum hydroxide,aluminum phosphate); saponins purified from the bark of the Q. saponariatree such as QS21 (a glycolipid that elutes in the 21st peak with HPLCfractionation; Antigenics, Inc., Worcester, Mass.);poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA), Flt3 ligand, Leishmania elongation factor (a purifiedLeishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS(immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvantsystem #4 which contains alum and MPL; SBB, Belgium), non-ionic blockcopolymers that form micelles such as CRL 1005 (these contain a linearchain of hydrophobic polyoxypropylene flanked by chains ofpolyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g.,IMS 1312, water-based nanoparticles combined with a solubleimmunostimulant, Seppic)

Adjuvants may be TLR ligands. Adjuvants that act through TLR3 includewithout limitation double-stranded RNA. Adjuvants that act through TLR4include without limitation derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton,Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland). Adjuvants that act through TLR5 includewithout limitation flagellin. Adjuvants that act through TLR7 and/orTLR8 include single-stranded RNA, oligoribonucleotides (ORN), syntheticlow molecular weight compounds such as imidazoquinolinamines (e.g.,imiquimod (R-837), resiquimod (R-848)). Adjuvants acting through TLR9include DNA of viral or bacterial origin, or syntheticoligodeoxynucleotides (ODN), such as CpG ODN. Another adjuvant class isphosphorothioate containing molecules to such as phosphorothioatenucleotide analogs and nucleic acids containing phosphorothioatebackbone linkages.

Immunoinhibitory Agents.

As used herein, an immunoinhibitory agent is an agent that inhibits animmune response in a subject to whom it is administered, whether aloneor in combination with another agent. Examples include steroids,retinoic acid, dexamethasone, cyclophosphamide, anti-CD3 antibody orantibody fragment, and other immunosuppressants.

Anti-Cancer Agents.

As used herein, an anti-cancer agent is an agent that at least partiallyinhibits the development or progression of a cancer, includinginhibiting in whole or in part symptoms associated with the cancer evenif only for the short term. Several anti-cancer agents can becategorized as DNA damaging agents and these include topoisomeraseinhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide,mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan,thiotepa, carmustine, lomustine, carboplatin, dacarbazine,procarbazine), DNA strand break inducing agents (e.g., bleomycin,doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubuleagents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g.,cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine,6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin,chlorodeoxyadenosine), anthracyclines, vinca alkaloids. orepipodophyllotoxins.

Examples of anti-cancer agents include without limitation Acivicin;Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin (aplatinum-containing regimen); Carmustine; Carubicin Hydrochloride;Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (aplatinum-containing regimen); Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin;Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel(TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone; Duazomycin;Edatrexate; Eflornithine; Elsamitrucin; Enloplatin; Enpromate;Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA), Esorubicin;Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine;Fenretinide; Floxuridine; Fludarabine; to 5-Fluorouracil;Fluorocitabine; Fosquidone; Fostriecin; Gefitinib (IRESSA), Gemcitabine;Hydroxyurea; Idarubicin; Ifosfamide; Ilmofosine; Imatinib mesylate(GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferonalpha-n1; Interferon alpha-n3; Interferon beta-I a; Interferon gamma-Ib; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLIMID, REVIMID);Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine; Losoxantrone;Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid;Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed(ALIMTA), Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin;Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate;Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin;Prednimustine; Procarbazine; Puromycin; Pyrazofurin; Riboprine;Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside; Sparfosate;Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin; Streptonigrin;Streptozocin; Sulofenur; Talisomycin; Tamsulosin; Taxol; Taxotere;Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR);Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) andderivatives thereof; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;Tirapazamine; Topotecan; Toremifene; Trestolone; Triciribine;Trimetrexate; Triptorelin; Tubulozole; Uracil Mustard; Uredepa;Vapreotide; Verteporfin; Vinblastine; Vincristine; Vindesine;Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine;Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin.

The anti-cancer agent may be an enzyme inhibitor including withoutlimitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinaseinhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may bewithout limitation Genistein (4′,5,7-trihydroxyisoflavone), Tyrphostin25 (3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, Herbimycin A,Daidzein (4′,7-dihydroxyisoflavone), AG-126,trans-1-(3′-carboxy-4′-hydroxyphenyl)-2-(2″,5″-dihydroxy-phenyl)ethane,or HDBA (2-Hydroxy-5-(2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid.The CDK inhibitor may be without limitation p21, p27, p57, p15, p16,p18, or p19. The MAP kinase inhibitor may be without limitation KY12420(C₂₃H₂₄O₈), CNI-1493, PD98059, or 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole. The EGFR inhibitor may be withoutlimitation erlotinib (TARCEVA), gefitinib (IRESSA), WH1-P97 (quinazolinederivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF,lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.

The anti-cancer agent may be a VEGF inhibitor including withoutlimitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib(MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA),anecortave (RETAANE), squalamine lactate, and semaphorin.

The anti-cancer agent may be an antibody or an antibody fragmentincluding without limitation an antibody or an antibody fragmentincluding but not limited to bevacizumab (AVASTIN), trastuzumab(HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chroniclymphocytic leukemia), gemtuzumab (MYLOTARG, hP67.6, anti-CD33,indicated for leukemia such as acute myeloid leukemia), rituximab(RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cellmalignancy), MDX-210 (bispecific antibody that binds simultaneously toHER-2/neu oncogene protein product and type I Fc receptors forimmunoglobulin G (IgG) (Fc gamma R1)), oregovomab (OVAREX, indicated forovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX),palivizumab (SYNAGIS, indicated for respiratory conditions such as RSVinfection), ibritumomab tiuxetan (ZEVALIN, indicated for Non-Hodgkin'slymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72),IOR-05, IOR-T6 (anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT OV103),epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H(indicated for brain cancer, melanoma).

Hematopoietic Differentiating Agents.

The agent may be one that stimulates the differentiation ofhematopoietic progenitor cells towards one or more lineages. Examplesinclude without limitation IL-3, G-CSF, GM-CSF, M-CSF, thrombopoeitin,erythropoietin, Wnt5A, Wnt11A, and the like.

Hematopoietic Self-Renewing Agents.

The agent may be one that stimulates the self-renewal of hematopoieticprogenitor cells. Examples include without limitation kit ligand,GSK3-beta inhibitors, Wnt5A together with SLF, Notch1 activators, Lnkinhibitors, prostaglandin E2 (PGE2) and agents that stimulate the PGE2pathway including PGE2, PGI2, Linoleic Acid, 13(s)-HODE, LY171883, MeadAcid, Eicosatrienoic Acid, Epoxyeicosatrienoic Acid, ONO-259, Cay1039, aPGE2 receptor agonist, of 16,16-dimethyl PGE2, 19(R)-hydroxy PGE2,16,16-dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester,11-deoxy-16,16-dimethyl PGE2,9-deoxy-9-methylene-16,16-dimethylPGE2,9-deoxy-9-methylene PGE2, Butaprost, Sulprostone, PGE2 serinolamide, PGE2 methyl ester, 16-phenyl tetranor PGE2,15(S)-15-methylPGE2,15 (R)-15-methyl PGE2, BIO, 8-bromo-cAMP, Forskolin, Bapta-AM,Fendiline, Nicardipine, Nifedipine, Pimozide, Strophanthidin,Lanatoside, L-Arg, Sodium Nitroprusside, Sodium Vanadate, Bradykinin,Mebeverine, Flurandrenolide, Atenolol, Pindolol, Gaboxadol, KynurenicAcid, Hydralazine, Thiabendazole, Bicuclline, Vesamicol, Peruvoside,Imipramine, Chlorpropamide, 1,5-Pentamethylenetetrazole,4-Aminopyridine, Diazoxide, Benfotiamine, 12-Methoxydodecenoic acid,N-Formyl-Met-Leu-Phe, Gallamine, IAA 94, Chlorotrianisene, andderivatives thereof, and the like.

Anti-Infective Agents.

The agent may be an anti-infective agent including without limitation ananti-bacterial agent, an anti-viral agent, an anti-parasitic agent, ananti-fungal agent, and an anti-mycobacterial agent.

Anti-bacterial agents may be without limitation β-lactam antibiotics,penicillins (such as natural penicillins, aminopenicillins,penicillinase-resistant penicillins, carboxy penicillins, ureidopenicillins), cephalosporins (first generation, second generation, andthird generation cephalosporins), other β-lactams (such as imipenem,monobactams), β-lactamase inhibitors, vancomycin, aminoglycosides andspectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin,clindamycin, rifampin, metronidazole, polymyxins, sulfonamides andtrimethoprim, or quinolines.

Other anti-bacterials may be without limitation Acedapsone; AcetosulfoneSodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil;Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; AmikacinSulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin;Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin;Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin;Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin;Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins;Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem;Biniramycin; Biphenamine Hydrochloride; Bispyrithione Magsulfex;Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox;Carbenicillin Disodium; Carbenicillin Indanyl Sodium; CarbenicillinPhenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefaclor;Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium;Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium;Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol;Cefixime; Cefinenoxime Hydrochloride; Cefmetazole; Cefmetazole Sodium;Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide;Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam toHydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; CefpimizoleSodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; CefpodoximeProxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime;Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime;Cefuroxime Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; CephacetrileSodium; Cephalexin; Cephalexin Hydrochloride; Cephaloglycin;Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine;Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol;Chloramphenicol Palmitate; Chloramphenicol Pantothenate Complex;Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; to Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfas alazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; orZorbamycin. Anti-mycobacterial agents may be without limitationMyambutol (Ethambutol Hydrochloride), Dapsone(4,4′-diaminodiphenylsulfone), Paser Granules (aminosalicylic acidgranules), Priftin to (rifapentine), Pyrazinamide, Isoniazid, Rifadin(Rifampin), Rifadin IV, Rifamate (Rifampin and Isoniazid), Rifater(Rifampin, Isoniazid, and Pyrazinamide), Streptomycin Sulfate orTrecator-SC (Ethionamide).

Anti-viral agents may be without limitation amantidine and rimantadine,ribivarin, acyclovir, vidarabine, trifluorothymidine, ganciclovir,zidovudine, retinovir, and interferons.

Anti-viral agents may be without limitation further include Acemannan;Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox;Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate;Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; DelavirdineMesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene;Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine;Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir;Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir;Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; SomantadineHydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride;Trifluridine; Valacyclovir Hydrochloride; Vidarabine; VidarabinePhosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine;Zidovudine; Zinviroxime or integrase inhibitors.

Anti-fungal agents may be without limitation imidazoles and triazoles,polyene macrolide antibiotics, griseofulvin, amphotericin B, andflucytosine. Antiparasites include heavy metals, antimalarialquinolines, folate antagonists, nitroimidazoles, benzimidazoles,avermectins, praxiquantel, ornithine decarboxylase inhibitors, phenols(e.g., bithionol, niclosamide); synthetic alkaloid (e.g.,dehydroemetine); piperazines (e.g., diethylcarbamazine); acetanilide(e.g., diloxanide furonate); halogenated quinolines (e.g., iodoquinol(diiodohydroxyquin)); nitrofurans (e.g., nifurtimox); diamidines (e.g.,pentamidine); tetrahydropyrimidine (e.g., pyrantel pamoate); or sulfatednaphthylamine (e.g., suramin). Other anti-infective agents may bewithout limitation Difloxacin Hydrochloride; Lauryl IsoquinoliniumBromide; Moxalactam Disodium; Ornidazole; Pentisomicin; SarafloxacinHydrochloride; Protease inhibitors of HIV and other retroviruses;Integrase Inhibitors of HIV and other retroviruses; Cefaclor (Ceclor);Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin (Mefoxin);Cefuroxime axetil (Ceftin); Ciprofloxacin (Cipro); AminacrineHydrochloride; Benzethonium Chloride: Bithionolate Sodium;Bromchlorenone; Carbamide Peroxide; Cetalkonium Chloride;Cetylpyridinium Chloride: Chlorhexidine Hydrochloride; Clioquinol; toDomiphen Bromide; Fenticlor; Fludazonium Chloride; Fuchsin, Basic;Furazolidone; Gentian Violet; Halquinols; Hexachlorophene: HydrogenPeroxide; Ichthammol; Imidecyl Iodine; Iodine; Isopropyl Alcohol;Mafenide Acetate; Meralein Sodium; Mercufenol Chloride; Mercury,Ammoniated; Methylbenzethonium Chloride; Nitrofurazone; Nitromersol;Octenidine Hydrochloride; Oxychlorosene; Oxychlorosene Sodium;Parachlorophenol, Camphorated; Potassium Permanganate; Povidone-Iodine;Sepazonium Chloride; Silver Nitrate; Sulfadiazine, Silver; Symclosene;Thimerfonate Sodium; Thimerosal; or Troclosene Potassium.

Nucleic Acid Agents.

Nucleic acids that can be delivered to a subject according to theinvention include naturally or non-naturally occurring DNA (includingcDNA, genomic DNA, nuclear DNA, mitochondrial DNA), RNA (including mRNA,rRNA, tRNA), oligonucleotides, a triple-helix forming molecule,immunostimulatory nucleic acids such as those described in U.S. Pat. No.6,194,388 (the teachings of which relating to immunostimulatory CpGnucleic acids are incorporated herein by reference), small interferingRNA (siRNA) or microRNAs (miRNA) used to modulate gene expression,antisense oligonucleotides used to modulate gene expression, aptamers,ribozymes, a gene or gene fragment, a regulatory sequence, includinganalogs, derivatives, and combinations thereof. These nucleic acids maybe administered neat or complexed to another entity, for example inorder to facilitate their binding to and/or uptake by target tissuesand/or cells.

Anti-Inflammatory Agents.

Anti-inflammatory agents are agents that reduce or eliminateinflammation. They include Alclofenac; Alclometasone Dipropionate;Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; AmfenacSodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen;Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; BenzydamineHydrochloride; Bromelains; Broperamole; Budesonide; Carprofen;C₁₋cloprofen; Cintazone; Cliprofen; Clobetasol Propionate; ClobetasoneButyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate;Cortodoxone; Deflazacort; Desonide; Desoximetasone; DexamethasoneDipropionate; Diclofenac Potassium; Diclofenac Sodium; DiflorasoneDiacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone;Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium;Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone;Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin;Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone;Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam;Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; TolmetinSodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids;Zomepirac Sodium. One preferred anti-inflammatory agent is aspirin.

Other Agents.

The agent may be without limitation adrenergic agent; adrenocorticalsteroid; adrenocortical suppressant; alcohol deterrent; aldosteroneantagonist; ammonia detoxicant; amino acid; amylotropic lateralsclerosis agent; anabolic; analeptic; analgesic; androgen; anesthetic;anorectic; anorexic; anterior pituitary activator; anterior pituitarysuppressant; anthelmintic; anti-acne agent; anti-adrenergic;anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti-anginal;anti-anxiety; anti-arthritic; anti-asthmatic including β-adrenergicagonists, methylxanthines, mast cell stabilizing agents,anticholinergics, adrenocortical steroids such as glucocorticoids;anti-atherosclerotic; anticholelithic; anticholelithogenic;anticholinergic; anticoagulant; anticoccidal; anticonvulsant;antidepressant; antidiabetic; antidiarrheal; antidiuretic; antidote;antidyskinetic; anti-emetic; anti-epileptic; anti-estrogen;antifibrinolytic; antiglaucoma; antihemorrhagic; antihemorrheologic;antihistamine; antihyperlipidemic; antihyperlipoproteinemic;antihypertensive; antihypotensive; anti-infective; anti-inflammatory;antikeratinizing agent; antimigraine; antimitotic; antimycotic;antinauseant; antineutropenic; antiobsessional agent; antioxidant;antiparkinsonian; antiperistaltic; antipneumocystic; antiprostatichypertrophy agent; antiprotozoal; antipruritic; antipsoriatic;antipsychotic; antirheumatic; antischistosomal; antiseborrheic;antisecretory; antispasmodic; antithrombotic; antitussive;anti-ulcerative; anti-urolithic; appetite suppressant; blood glucoseregulator; bone resorption inhibitor; bronchodilator; carbonic anhydraseinhibitor; cardiac depressant; cardioprotectant; cardiotonic;cardiovascular agent; cerebral ischemia agent; choleretic; cholinergic;cholinergic agonist; cholinesterase deactivator; coccidiostat; cognitionadjuvant; cognition enhancer; conjunctivitis agent; contrast agent;depressant; diagnostic aid; diuretic; dopaminergic agent;ectoparasiticide; emetic; enzyme inhibitor; estrogen; estrogen receptoragonist; fibrinolytic; fluorescent agent; free oxygen radical scavenger;gastric acid suppressant; gastrointestinal motility effector; geriatricagent; glucocorticoid; gonad-stimulating principle; hair growthstimulant; hemostatic; herbal active agent; histamine H2 receptorantagonists; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic;hypotensive; HMGCoA reductase inhibitor; impotence therapy adjunct;inflammatory bowel disease agent; keratolytic; LHRH agonist; liverdisorder agent; luteolysin; memory adjuvant; mental performanceenhancer; mineral; mood regulator; mucolytic; mucosal protective agent;multiple sclerosis agent; mydriatic; nasal decongestant; neuroleptic;neuromuscular blocking agent; neuroprotective; NMDA antagonist;non-hormonal sterol derivative; nutrient; oxytocic; Paget's diseaseagent; plasminogen activator; platelet activating factor antagonist;platelet aggregation inhibitor; post-stroke and post-head trauma agents;progestin; prostaglandin; prostate growth inhibitor; prothyrotropin;psychotropic; radioactive agent; relaxant; rhinitis agent; scabicide;sclerosing agent; sedative; sedative-hypnotic; selective adenosine A1antagonist; sequestering agents; serotonin antagonist; serotonininhibitor; serotonin receptor antagonist; steroid; stimulant;suppressant; thyroid hormone; thyroid inhibitor; thyromimetic;tranquilizer; unstable angina agent; uricosuric; vasoconstrictor;vasodilator; vulnerary; wound healing agent; or xanthine oxidaseinhibitor.

Subjects

The invention can be practiced in virtually any subject type that islikely to benefit from delivery of agents as contemplated herein. Humansubjects are preferred subjects in some embodiments of the invention.Subjects also include animals such as household pets (e.g., dogs, cats,rabbits, ferrets, etc.), livestock or farm animals (e.g., cows, pigs,sheep, chickens and other poultry), horses such as thoroughbred horses,laboratory animals (e.g., mice, rats, rabbits, etc.), and the like.Subjects also include fish and other aquatic species.

The subjects to whom the agents are delivered may be normal subjects.Alternatively they may have or may be at risk of developing a conditionthat can be diagnosed or that can to benefit from delivery of one ormore particular agents.

Such conditions include cancer (e.g., solid tumor cancers or non-solidcancer such as leukemias), infections (including infections localized toparticular regions or tissues in the body), autoimmune disorders,allergies or allergic conditions, asthma, transplant rejection, and thelike.

Tests for diagnosing various of the conditions embraced by the inventionare known in the art and will be familiar to the ordinary medicalpractitioner. These laboratory tests include without limitationmicroscopic analyses, cultivation dependent tests (such as cultures),and nucleic acid detection tests. These include wet mounts,stain-enhanced microscopy, immune microscopy (e.g., FISH), hybridizationmicroscopy, particle agglutination, enzyme-linked immunosorbent assays,urine screening tests, DNA probe hybridization, serologic tests, etc.The medical practitioner will generally also take a full history andconduct a complete physical examination in addition to running thelaboratory tests listed above.

A subject having a cancer is a subject that has detectable cancer cells.A subject at risk of developing a cancer is a subject that has a higherthan normal probability of developing cancer. These subjects include,for instance, subjects having a genetic abnormality that has beendemonstrated to be associated with a higher likelihood of developing acancer, subjects having a familial disposition to cancer, subjectsexposed to cancer causing agents (i.e., carcinogens) such as tobacco,asbestos, or other chemical toxins, and subjects previously treated forcancer and in apparent remission.

Subjects having an infection are those that exhibit symptoms thereofincluding without limitation fever, chills, myalgia, photophobia,pharyngitis, acute lymphadenopathy, splenomegaly, gastrointestinalupset, leukocytosis or leukopenia, and/or those in whom infectiouspathogens or byproducts thereof can be detected.

A subject at risk of developing an infection is one that is at risk ofexposure to an infectious pathogen. Such subjects include those thatlive in an area where such pathogens are known to exist and where suchinfections are common. These subjects also include those that engage inhigh risk activities such as sharing of needles, engaging in unprotectedsexual activity, routine contact with infected samples of subjects(e.g., medical practitioners), people who have undergone surgery,including but not limited to abdominal surgery, etc.

The subject may have or may be at risk of developing an infection suchas a bacterial to infection, a viral infection, a fungal infection, aparasitic infection or a mycobacterial infection. In these embodiments,the vesicles may comprise an anti-microbial agent such as ananti-bacterial agent, an anti-viral agent, an anti-fungal agent, ananti-parasitic agent, or an anti-mycobacterial agent and the cellcarriers (e.g., the T cells) may be genetically engineered to produceanother agent useful in stimulating an immune response against theinfection, or potentially treating the infection.

Cancer

The invention contemplates administration of agents to subjects havingor at risk of developing a cancer including for example a solid tumorcancer, using the vesicles of the invention. The agents may beanti-cancer agents, including chemotherapeutics, antibody basedtherapeutics, hormone based therapeutics, and enzyme inhibitory agents,and/or they may be immunostimulatory agents such as antigens (e.g.,cancer antigens) and/or adjuvants, and/or they may be diagnostic agents(e.g., imaging agents), or any of the other agents described herein. Theinvention contemplates that the vesicles of the invention are able todeliver higher quantities of these agents, alone or in combination, tothese subjects, and/or to allow prolonged exposure of the subject tothese agents via a slow steady release profile.

The cancer may be carcinoma, sarcoma or melanoma. Carcinomas includewithout limitation to basal cell carcinoma, biliary tract cancer,bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNScancer, colon and rectum cancer, kidney or renal cell cancer, larynxcancer, liver cancer, small cell lung cancer, non-small cell lung cancer(NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma, andsquamous cell carcinoma), oral cavity cancer, ovarian cancer, pancreaticcancer, prostate cancer, skin cancer (including basal cell cancer andsquamous cell cancer), stomach cancer, testicular cancer, thyroidcancer, uterine cancer, rectal cancer, cancer of the respiratory system,and cancer of the urinary system.

Sarcomas are rare mesenchymal neoplasms that arise in bone(osteosarcomas) and soft tissues (fibrosarcomas). Sarcomas includewithout limitation liposarcomas (including myxoid liposarcomas andpleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas,malignant peripheral nerve sheath tumors (also called malignantschwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors(including Ewing's sarcoma of bone, extraskeletal (i.e., not bone)Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma,angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma,to hemangioendothelioma, desmoid tumor (also called aggressivefibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibroushistiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolarsoft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplasticsmall cell tumor, gastrointestinal stromal tumor (GIST) (also known asGI stromal sarcoma), and chondrosarcoma.

Melanomas are tumors arising from the melanocytic system of the skin andother organs. Examples of melanoma include without limitation lentigomaligna melanoma, superficial spreading melanoma, nodular melanoma, andacral lentiginous melanoma. The cancer may be a solid tumor lymphoma.Examples include Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B celllymphoma.

The cancer may be without limitation bone cancer, brain cancer, breastcancer, colorectal cancer, connective tissue cancer, cancer of thedigestive system, endometrial cancer, esophageal cancer, eye cancer,cancer of the head and neck, gastric cancer, intra-epithelial neoplasm,melanoma neuroblastoma, Non-Hodgkin's lymphoma, non-small cell lungcancer, prostate cancer, retinoblastoma, or rhabdomyosarcoma.

Infection

The invention contemplates administration of agents to subjects havingor at risk of developing an infection such as a bacterial infection, aviral infection, a fungal infection, a parasitic infection or amycobacterial infection, using the vesicles of the invention. The agentsmay be anti-infective agents including anti-bacterial agents, anti-viralagents, anti-fungal agents, anti-parasitic agents, andanti-mycobacterial agents), immunostimulatory agents such as antigens(e.g., microbial antigens such as bacterial antigens, viral antigens,fungal antigens, parasitic antigens, and mycobacterial antigens) and/oradjuvants, diagnostic agents (e.g., imaging agents), or any of the otheragents described herein. The invention contemplates that the vesicles ofthe invention are able to deliver higher quantities of these agents,alone or in combination, to these subjects, and/or to allow prolongedexposure of the subject to these agents via a slow steady releaseprofile.

The bacterial infection may be without limitation an E. coli infection,a Staphylococcal infection, a Streptococcal infection, a Pseudomonasinfection, Clostridium difficile infection, Legionella infection,Pneumococcus infection, Haemophilus infection, Klebsiella infection,Enterobacter infection, Citrobacter infection, Neisseria infection,Shigella infection, Salmonella to infection, Listeria infection,Pasteurella infection, Streptobacillus infection, Spirillum infection,Treponema infection, Actinomyces infection, Borrelia infection,Corynebacterium infection, Nocardia infection, Gardnerella infection,Campylobacter infection, Spirochaeta infection, Proteus infection,Bacteriodes infection, H. pylori infection, or anthrax infection.

The mycobacterial infection may be without limitation tuberculosis orleprosy respectively caused by the M. tuberculosis and M. lepraespecies.

The viral infection may be without limitation a Herpes simplex virus 1infection, a Herpes simplex virus 2 infection, cytomegalovirusinfection, hepatitis A virus infection, hepatitis B virus infection,hepatitis C virus infection, human papilloma virus infection, EpsteinBarr virus infection, rotavirus infection, adenovirus infection,influenza virus infection, influenza A virus infection, H1N1 (swine flu)infection, respiratory syncytial virus infection, varicella-zoster virusinfections, small pox infection, monkey pox infection, SARS infection oravian flu infection.

The fungal infection may be without limitation candidiasis, ringworm,histoplasmosis, blastomycosis, paracoccidioidomycosis, crytococcosis,aspergillosis, chromomycosis, mycetoma infections, pseudallescheriasis,or tinea versicolor infection.

The parasite infection may be without limitation amebiasis, Trypanosomacruzi infection, Fascioliasis, Leishmaniasis, Plasmodium infections,Onchocerciasis, Paragonimiasis, Trypanosoma brucei infection,Pneumocystis infection, Trichomonas vaginalis infection, Taeniainfection, Hymenolepsis infection, Echinococcus infections,Schistosomiasis, neurocysticercosis, Necator americanus infection, orTrichuris trichuria infection.

Allergy and Asthma

The invention contemplates administration of agents to subjects havingor at risk of developing an allergy or asthma. The agents may beallergens, immunostimulatory agents including agents that stimulate aTh1 response, immunoinhibitory or immunosuppressant agents includingagents that inhibit a Th2 response, anti-inflammatory agents,leukotriene antagonists, IL-4 muteins, soluble IL-4 receptors, anti-IL-4antibodies, IL-4 antagonists, anti-IL-5 antibodies, soluble IL-13receptor-Fc fusion proteins, anti-IL-9 antibodies, CCR3 antagonists,CCR5 antagonists, VLA-4 inhibitors, and other downregulators of IgE suchas but not limited to anti-IgE, cytokines such as Th1 cytokines such asIL-12 and IFN-gamma, steroids including to corticosteroids such asprednisolone, and/or they may be diagnostic agents (e.g., imagingagents), or any of the other agents described herein. The inventioncontemplates that the vesicles of the invention are able to deliverhigher quantities of these agents, alone or in combination, to thesesubjects, and/or to allow prolonged exposure of the subject to theseagents via a slow steady release profile.

An allergy is an acquired hypersensitivity to an allergen. Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, hay fever, bronchial asthma, urticaria (hives) and foodallergies, and other atopic conditions. Allergies are generally causedby IgE antibody generation against harmless allergens. Asthma is adisorder of the respiratory system characterized by inflammation,narrowing of the airways and increased reactivity of the airways toinhaled agents. Asthma is frequently, although not exclusively,associated with atopic or allergic symptoms.

Autoimmune Disease

The invention contemplates administration of agents to subjects havingor at risk of developing an autoimmune disease or disorder. The agentsmay be immunoinhibitory or immunosuppressant agents including those thatinhibit a Th1 response, immunostimulatory agents that stimulate a Th2response, cytokines such as IL-4, IL-5 and IL-10, anti-inflammatoryagents, and/or they may be diagnostic agents (e.g., imaging agents), orany of the other agents described herein. The invention contemplatesthat the vesicles of the invention are able to deliver higher quantitiesof these agents, alone or in combination, to these subjects, and/or toallow prolonged exposure of the subject to these agents via a slowsteady release profile.

Autoimmune disease is a class of diseases in which a subject's ownantibodies react with host tissue or in which immune effector T cellsare autoreactive to endogenous self peptides and cause destruction oftissue. Thus an immune response is mounted against a subject's ownantigens, referred to as self antigens. Autoimmune diseases aregenerally considered to be Th1 biased. As a result, induction of a Th2immune response or Th2 like cytokines can be beneficial.

Autoimmune diseases include but are not limited to rheumatoid arthritis,Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE),autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto'sthyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigusvulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, scleroderma with anti-collagen antibodies,mixed connective tissue disease, polymyositis, pernicious anemia,idiopathic Addison's disease, autoimmune-associated infertility,glomerulonephritis (e.g., crescentic glomerulonephritis, proliferativeglomerulonephritis), bullous pemphigoid, Sjögren's syndrome, insulinresistance, and autoimmune diabetes mellitus.

Transplant Therapy

The invention contemplates administration of agents to subjectsundergoing a cell or organ transplant. The agents may beimmunoinhibitory or immunosuppressant agents, anti-inflammatory agents,and/or they may be diagnostic agents (e.g., imaging agents), or any ofthe other agents described herein. The invention contemplates that thevesicles of the invention are able to deliver higher quantities of theseagents, alone or in combination, to these subjects, and/or to allowprolonged exposure of the subject to these agents via a slow steadyrelease profile.

The compositions and methods provided herein may also be used tomodulate immune responses following transplant therapy. Transplantsuccess is often limited by rejection of the transplanted tissue by thebody's immune system. As a result, transplant recipients are usuallyimmunosuppressed for extended periods of time in order to allow thetransplanted tissue to survive. The invention contemplates delivery ofimmunomodulators, and particularly immunoinhibitory agents, totransplant sites in order to minimize transplant rejection. Thus, theinvention contemplates administration to subjects that are going toundergo, are undergoing, or have undergone a transplant.

The foregoing lists are not intended to be exhaustive but ratherexemplary. Those of ordinary skill in the art will identify otherexamples of each condition type that are amenable to prevention andtreatment using the methods of the invention.

Effective Amounts, Regimens, Formulations

The agents are administered in the form of stabilized MLVs and ineffective amounts. An effective amount is a dosage of the agentsufficient to provide a medically desirable result. The effective amountwill vary with the desired outcome, the particular condition beingtreated to or prevented, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent or combination therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. It is preferredgenerally that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment.

For example, if the subject has a tumor, an effective amount may be thatamount that reduces the tumor volume or load (as for example determinedby imaging the tumor). Effective amounts may also be assessed by thepresence and/or frequency of cancer cells in the blood or other bodyfluid or tissue (e.g., a biopsy). If the tumor is impacting the normalfunctioning of a tissue or organ, then the effective amount may beassessed by measuring the normal functioning of the tissue or organ.

In some instances the effective amount is the amount required to lessenor eliminate one or more, and preferably all, symptoms. For example, ina subject having an allergy or experiencing an asthmatic attack, aneffective amount of an agent may be that amount that lessens oreliminates the symptoms associated with the allergy or the asthmaticattack. They may include sneezing, hives, nasal congestion, and laboredbreathing. Similarly, in a subject having an infection, an effectiveamount of an agent may be that amount that lessens or eliminate thesymptoms associated with the infection. These may include fever andmalaise. If the agent is a diagnostic agent, an effective amount may bean amount that allows visualization of the body region or cells ofinterest. If the agent is an antigen, the effective amount may be thatamount that triggers an immune response against the antigen andpreferably provides short and even more preferably long term protectionagainst the pathogen from which the antigen derives. It will beunderstood that in some instances the invention contemplates singleadministration of an agent and in some instances the inventioncontemplates multiple administrations of an agent. As an example, anantigen may be administered in a prime dose and a boost dose, althoughin some instances the invention provides sufficient delivery of theantigen, and optionally an adjuvant, that no boost dose is required.

The invention provides pharmaceutical compositions. Pharmaceuticalcompositions are sterile compositions that comprise the vesicles of theinvention and preferably agent(s), preferably in apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other subject contemplated bythe invention.

The term “carrier” denotes an organic or inorganic ingredient, naturalor synthetic, with which vesicles and preferably agent(s) are combinedto facilitate administration. The components of the pharmaceuticalcompositions are commingled in a manner that precludes interaction thatwould substantially impair their desired pharmaceutical efficiency.

Suitable buffering agents include acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); and parabens (0.01-0.25% W/V).

Unless otherwise stated herein, a variety of administration routes areavailable. The particular mode selected will depend, of course, upon theparticular active agent selected, the particular condition being treatedand the dosage required for therapeutic efficacy. The methods provided,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of a desired response without causing clinically unacceptableadverse effects. One mode of administration is a parenteral route. Theterm “parenteral” includes subcutaneous injections, intravenous,intramuscular, intraperitoneal, intra sternal injection or infusiontechniques. Other modes of administration include oral, mucosal, rectal,vaginal, sublingual, intranasal, intratracheal, inhalation, ocular,transdermal, etc.

For oral administration, the compounds can be formulated readily bycombining the vesicles with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable formulation as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, films, suspensionsand the like, for oral ingestion by a subject to be treated. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). Optionally the oral formulations may also beformulated in saline or buffers for neutralizing internal acidconditions or may be administered without any carriers.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the vesicles suspended in suitable liquids, such asaqueous solutions, buffered solutions, fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount.

When it is desirable to deliver the compositions of the inventionsystemically, they may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers. Pharmaceutical parenteral formulations includeaqueous solutions of the ingredients. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively,suspensions of vesicles may be prepared as oil-based suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Alternatively, the vesicles may be in powder form or lyophilized formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The compositions may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In Vitro Use

The invention further contemplates in vitro applications such as cellculturing and tissue engineering, that require or for which it would bemore convenient to have a constant source of one or more agents such asbut not limited to cell growth factors, and the like.

Kits

The invention further contemplates kits comprising the vesicles of theinvention. The vesicles may comprise one or more agents of interest. Thekits may further comprise one or more agents of interest to beincorporated into the vesicles. These kits may also include writtenmaterials such as instructions for use of the vesicles. The vesicles maybe provided in a buffer or in a lyophilized form, preferably with asucrose-containing excipient.

The invention also contemplates kits comprising the various substrates,reagents and catalysts required for synthesizing the vesicles of theinvention. Such kits may include for example lipids such as thosedescribed herein, functionalized components of a lipid bilayer such asfunctionalized lipids, one or more crosslinkers such as membranepermeable crosslinkers, multivalent cations such as divalent cations,and the like. These kits may also include written materials such asinstructions for synthesizing the vesicles. The kits may also includethe agents of interest.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

To address limitations in prior art methods, we envisioned the synthesisof lipid particles stabilized by forming crosslinks connecting theheadgroups of adjacent lipid layers within multilamellar vesicles(MLVs). Divalent cations are known to induce fusion of liposomes intoMLVs. (See Duzgunes et al., J Membrane Biol. 1981; 59:115-125.) Wemodified this process by introducing maleimide-functionalized lipids(e.g., MPB) into vesicles and crosslinking layers of MPB usingdithiol-crosslinkers (e.g., synthesis schematics shown in FIG. 1A). Theresulting interbilayer-crosslinked multilamellar lipid vesicles (ICMVs)exhibited attractive features, such as greatly enhanced proteinencapsulation efficiency (100-fold relative to simple liposomes, FIG.5), protein loading per mass of particles (20-fold relative to simpleliposomes, FIG. 5), and sustained retention of entrapped cargos in thepresence of serum with slow, sustained release kinetics (FIG. 6). Thesynthesis is carried entirely in aqueous buffers friendly to theentrapment of fragile protein cargos, and generates particles composedonly of biodegradable lipids. As a test-bed for biomedical applicationsof this new class of particles, we examined ICMVs as a delivery vehiclefor antigens and adjuvants. Specifically, we used ovalbumin (OVA) as amodel to antigen and tested co-delivery of toll-like receptor agonists(TLRa) in vivo for vaccine applications.

Materials and Methods

Lipids (DOPC:DOPG:maleimide-functionalized lipids in molar ratios of40:10:50) were dried to form lipid films, and rehydrated in 10 mMbis-tris propane (BTP) at pH 7.0 for 1 hr in the presence of cargomolecules. As shown in FIG. 1A, the resulting liposomes were sonicatedand induced to undergo fusion by addition of divalent cations such asMg²⁺ and Ca²⁺. The resulting MLVs were incubated with DTT (maleimide:DTTratio of 2:1) to conjugate apposing layers of maleimide-functionalizedlipids and form crosslinked ICMVs. The resulting structures werecentrifuged, washed, and then PEGylated by incubation with 2 kDaPEG-thiol for 30 min. The final products were centrifuged and washed 3×with deionized water. Particle size and surface charge was determined bydynamic light scattering (DLS) using a 90Plus particle size analyzer(Brookhaven Instruments). The particles were analyzed with confocalmicroscopy and cryo-TEM. To quantify the fraction of lipids exposed onthe external surfaces of particles, lamellarity assay was performed asdescribed previously. (See Girard et al., Biophysical journal 2004;87:419-429.) For encapsulation studies, ovalbumin (OVA) modified withalexa-fluor 555 were used to measure the amount encapsulated inPEGylated liposomes, lipid-coated PLGA particles (as previouslydescribed by Bershteyn et al., Soft Matter 2008; 4:1787-1791), andICMVs. SIV-Gag and FLT-3 ligand modified with alexa-fluor 555 were usedin some assays. Release of OVA from these particles was performed indialysis membranes with MW cutoff of 100 kDa.

For in vitro dendritic cell (DC) activation studies, bone marrow-derivedor splenic DCs were incubated with ICMVs encapsulating OVA incombination with monophorsphoryl lipid A (MPLA) and R-848, TLR4 and TLR7agonists, respectively. Cells were stained and analyzed by flowcytometry to examine the extent of DC activation and particleinternalization. The culture media were analyzed for IL12p70 expressionby ELISA (R&D Systems). For analysis of the in vivo function of ICMVs asvaccine carriers, groups of C57B1/6 mice were immunized s.c. in theflank with either 70 μg of OVA delivered in bolus injection or in ICMVs(with or without TLR agonists) and boosted 21 days later. Frequencies ofOVA-specific T-cells and interferon-gamma producing T-cells elicited byimmunization were determined by flow cytometry analysis of to peripheralblood mononuclear cells. Anti-OVA titers were determined by ELISAanalysis of sera from immunized mice. Animals were cared for followingNIH, state, and local guidelines.

Results and Discussion

The traditional protocol for forming MLVs was modified to synthesizestable ICMVs (FIG. 1A). First, liposomes containing up to 50 mol % MPBwere formed by rehydrating lipid films with sonication in the presenceof protein or drug cargo solutions. Fusion among the resulting liposomeswas induced by the addition of divalent cations to form MLVs as reportedpreviously. (See Duzgunes et al., J Membrane Biol. 1981; 59:115-125). Wethen introduced DTT as a membrane-permeable reagent to form covalentcrosslinks between maleimide headgroups of apposing lipid membraneswithin the MLVs (FIG. 1B). As shown in Tables 1A and 1B, both divalentcation and DTT were required to form stable ICMVs with significant yieldgreater than 40%; MLVs formed with either of Mg²⁺ or DTT alone did notproduce significant amount of lipid particles that were centrifuged with10K×g. The presence of 20 mM sodium chloride interfered with particleformation, as monovalent cation is known to inhibit fusion of vesiclesmediated by divalent cations. (See Duzgunes et al., J Membrane Biol.1981; 59:115-125.) At least 25 mol % or higher MPB were required toachieve a significant yield of particles, and we subsequently chose towork with a lipid composition of DOPC:DOPG:MPB in 40:10:50 molar ratio.

The resulting ICMVs had typical mean diameters of 250±40 nm withpolydispersity indices of ˜0.08 (FIG. 3A). CryoEM images of theparticles revealed multilamellar structures comprised of electron densebands thicker than single lipid bilayers, suggesting multilayers oflipids conjugated across the lipid membranes (FIG. 3B). The fraction oflipids displayed on the external surfaces of particles was measured withlamellarity assay. Sonicated liposomes had 37±2.3% of lipids on theexternal surface, whereas the value decreased to 19±1.0% in ICMVs formedafter modifying the liposomes with Mg²⁺ and DTT, suggesting theirmultilayered structures. The ICMVs were PEGylated by reactingsurface-displayed MPB with PEG-thiol (2 kDa MW). PEGylation increasedthe diameter of ICMVs to 272±40 nm, and the particles stored in PBS for7 days maintained their original sizes (Table 2, and some data notshown). The ICMVs were amenable to lyophilization with 3% sucrose addedas an excipient as the particles resuspended in buffered solution afterlyophilization maintained their overall morphologies and sizecharacteristics (Table 2).

Liposomes and PLGA particles have been widely used for vehicles ofprotein delivery; therefore, we compared protein encapsulation in thesevehicles to ICMVs. As shown in FIG. 5, ICMVs exhibited superior OVAencapsulation efficiency compared to traditional PEGylated stealthliposomes and lipid-coated PLGA particles (108 and 4 fold increases,respectively). Similarly, the amount of OVA encapsulated per totalparticle mass was enhanced in ICMVs by 21 and 10 fold, respectively. Wenext examined the amount of various proteins encapsulated in ICMV overthe course of its synthesis (FIG. 4A). The amount of SIV-gag, FLT-3ligand, and OVA encapsulated progressively increased from sonicatedliposomes, MLVs formed by Mg²⁺-mediated fusion, to ICMVs formed by Mg²⁺and DTT. In particular, more than 70% of OVA initially loaded wasencapsulated in the ICMVs at 407.5±18.7 ng OVA per mg of lipid. UsingOVA as a model protein, cargo release from these vesicles was compared(FIG. 6). OVA was released from ICMVs in a slow, continuous manner,whereas OVA was burst-released from liposomes and MLVs in media withserum.

Using OVA as a model vaccine antigen, we examined ICMVs as a platformfor vaccine delivery. ICMVs were loaded with OVA and Toll-like receptoragonists, MPLA (TLR4 ligand) and/or resiquimod (TLR7/8 ligand).Dendritic cells (DCs) incubated with OVA-loaded ICMVs in vitro avidlyendocytosed the particles. OVA-loaded ICMVs alone did not activate DCsin vitro, but particles bearing TLR agonists (TLRa) triggeredupregulation of costimulatory molecules (e.g. CD40, CD80, CD86, and MHCII), and IL-12p70 secretion by DCs in vitro; particles carrying bothTLR4 and TLR7 agonists promoted synergistic DC activation (FIG. 8).

To test the efficacy of particles co-delivering TLRa in combinationswith OVA for vaccination in vivo, C57B1/6 mice were immunized withOVA-loaded ICMVs and compared to the equivalent doses of soluble OVA.Interestingly, compared to the bolus injection of OVA, OVA-loaded ICMVsenhanced expansion of antigen-specific CD8⁺ T-cells by 17-fold, reaching˜7% among all CD8⁺ T-cells after boost (FIG. 10). These OVA-loadedparticles also elicited the highest frequency of functionally-competentinterferon-gamma (IFN-gamma)-producing OVA-specific T-cells. Notably,however, antibody responses against OVA required the presence of TLRafor maximal titers, and OVA-loaded ICMVs co-encapsulated with TLR4 andTLR7 agonists elicited ˜10-fold higher anti-OVA antibody titers comparedto the same doses of soluble OVA/TLRa (FIG. 10B).

To begin to understand the immune responses that ICMVs can elicitagainst antigens from clinically-relevant pathogens, we testedimmunization with an antigen from an infectious to agent. Various dosesof ICMVs having the antigen encapsulated therein and also having MPLAincorporated into their bilayers were used to immunize mice. After primeand boost regimen, strong antibody titers were generated against theantigen as detected in sera from day 34, whereas the equivalent dosesdelivered by soluble antigen and MPLA elicited much weaker antibodytiters (data not shown). These results suggest ICMVs as an effectivevehicle for delivering antigens for immunizations.

In this study, we developed novel multilamellar lipid particles thatexhibit attractive features: Protein encapsulation is greatly enhancedin the ICMVs without the use of organic solvents. These particlesexhibit colloidal stability, and the lipid-based particles are fullybiodegradable. We demonstrated the versatility of ICMVs as a platformfor vaccine delivery. These studies suggest that ICMVs should be broadlyuseful in a variety of in vivo drug delivery applications.

Example 2

The following Example is a more comprehensive description and reportingof some of the experiments and results described in Example 1.

Currently licensed vaccine adjuvants (e.g., aluminum hydroxide and theoil-in-water emulsion MF59) promote immunity by primarily elicitinghumoral immune responses, without stimulating cellular immunity^(1,2).As strong CD8⁺ T cell (CD8T) responses may be required for vaccinesagainst cancer or intracellular pathogens such as HIV, malaria, andhepatitis C, there is great interest in technologies to promoteconcerted humoral and cellular immune responses^(3,4). To this end,engineered live vaccine vectors such as non-replicating recombinantviruses have been developed⁵⁻⁷, which can induce both robust antibodyresponses and massive expansion of functional antigen-specific CD8⁺T-cells in murine models. However, safety concerns with live vectors andanti-vector immunity can complicate live vector vaccine design⁷.Pre-existing vector-specific immune responses have reduced theimmunogenicity of live vector-based vaccines in clinical trials⁸, andthe immune response raised against live vectors following a primingimmunization can render booster immunizations using the same vectorproblematic⁷.

In contrast, non-living synthetic vaccines delivering defined antigenscan be rationally designed to avoid anti-vector immunity⁹. Such“subunit” vaccines are composed of one or a few selected recombinantproteins or polysaccharides normally present in the structure of thetarget pathogen. However, subunit vaccines elicit poor or non-existentCD8T responses, due to the to low efficiency of cross-presentation (theuptake and processing of extracellular antigen by immune cells forpresentation on class I MHC molecules to naive CD8⁺ T-cells)¹⁰. Topromote cross-presentation, synthetic particles loaded with proteinantigens and defined immunostimulatory molecules have been used¹¹⁻¹⁷,mimicking in a reductionist fashion the cues provided to the immunesystem during infection by pathogens. Liposomes are particularlyattractive materials for this application, due to their low toxicity andimmunogenicity, track record of safety in clinical use, ease ofpreparation, and proven manufacturability at commercial scales^(18,19).Lipid vesicles in the form of unilamellar, multilamellar, or polymerizedvesicles have been tested as vaccine delivery materials, with somesuccess¹⁹⁻²³. Antigens entrapped in lipid vesicles are cross-presentedin vivo^(19,24,25), and liposomal protein vaccines have been shown toelicit protective T-cell-mediated anti-microbial and anti-tumor immuneresponses in small-animal models^(23,26,27). However, for diseases suchas HIV and cancer, it is currently believed that extremely potent T-cellresponses (in concert with humoral immunity) will be required to controlthe virus/tumors, and therefore, more potent T-cell vaccines are stillsought^(3,4).

A potential factor influencing the potency of lipid vesicles in vaccinedelivery is their limited stability in the presence of serum components.For liposomal cargos that can be processed at high temperature or loadedby diffusion through pre-formed vesicle membranes, enhanced vesiclestability can be achieved by using high-T_(m) lipids, especially whencombined with cholesterol and/or PEGylation²⁸. Uni- and multi-lamellarvesicles have also been stabilized by polymerizing reactive headgroupsat the surface of bilayers²⁹, polymerizing reactive groups inphosholipid acyl tails^(20,29), or polymerizing hydrophobic monomersadsorbed into the hydrophobic interior of membranes³⁰. Common to each ofthese approaches is the concept of polymerizing components in the planeof the bilayer. However, finding polymerization chemistries that can becarried out in mild conditions compatible with vaccine antigens ischallenging²⁰.

Here we describe a new class of lipid drug carriers,interbilayer-crosslinked multilamellar vesicles (ICMVs), formed bystabilizing multilamellar vesicles with short covalent crosslinkslinking lipid headgroups across the apposing faces of adjacenttightly-stacked bilayers within the vesicle walls. ICMVs encapsulatedand stably retained high levels of proteins, releasing entrapped cargovery slowly when exposed to serum (over 30 days) compared to simpleliposomes or multilamellar vesicles (MLVs) of the same lipidcomposition. However, these vesicles were quickly degraded in thepresence of lipases normally found at high levels within intracellularcompartments³¹. Using this novel vesicle structure to co-entrap highlevels of a model protein antigen (ovalbumin, OVA) and a lipid-likeimmunostimulatory ligand (monophosphoryl lipid A, MPLA), we carried outimmunization studies in mice and found that ICMVs elicited robustantibody titers ˜1000-fold greater than simple liposomes and ˜10-foldgreater than MLVs of identical lipid compositions. Unlike live vectorsthat are often only effective for a single injection administered to avector-naïve individual, these synthetic vesicles triggered steadilyincreasing humoral and CD8⁺ T-cell responses following repeatedadministrations, with antigen-specific T-cells expanding to a peak ofnearly 30% of the total CD8⁺ T-cells in blood following a prime and twobooster immunizations. These new materials may thus open the door tosubunit vaccines that are both safe and highly effective for generatingboth humoral and cellular immunity.

Materials and Methods

Synthesis of ICMVs. 1.26 μmol of lipids in chloroform (typical lipidcomposition:DOPC(1,2-Dioleoyl-sn-Glycero-3-Phosphocholine):DOPG(1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1′-rac-glycerol)):MPB(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide)=4:1:5 molar ratio, all lipids from Avanti Polar Lipids,Alabaster, Ala.) were dispensed to glass vials, and the organic solventswere evaporated under vacuum overnight to prepare dried thin lipidfilms. The lipid films were rehydrated in 10 mM bis-tris propane (BTP)at pH 7.0 with cargo proteins for 1 hr with rigorous vortexing every 10mM, and then sonicated in alternating power cycles of 6 watts and 3watts in 30 s intervals for 5 mM on ice (Misonix Microson XL probe tipsonicator, Farmingdale, N.Y.). The liposomes formed in this first stepwere induced to undergo fusion by addition of divalent cations such asMg²⁺ and Ca²⁺ at a final concentration of 10 mM. The resulting MLVs wereincubated with 1.5 mM DTT (maleimide:DTT molar ratio of 2:1) for 1 hr at37° C. to conjugate opposing bilayers of maleimide-functionalized lipidsand form crosslinked ICMVs; the resulting vesicles were recovered bycentrifugation at 14,000×g for 4 mM, and washed twice with deionizedwater. For PEGylation, the particles were incubated with 2 kDa PEG-thiol(Laysan Bio, Arab, Ala.) in a 1.5-fold molar excess of PEG-SH tomaleimide groups for 1 hr at 37° C. The resulting particles werecentrifuged and washed 3× with deionized water. The final products wereeither stored in PBS at 4° C. or lyophilized in the presence of 3%sucrose as a cryoprotectant and stored at −20° C. For some assays,simple liposomes or Mg-fused MLVs were harvested prior to crosslinkingwith to ultracentrifugation at 115K g using an Optima ultracentrifugefor 6 hrs (Beckman Coulter).

In vitro protein loading and drug release. For encapsulation studies,ovalbumin (OVA, Worthington, Lakewood, N.J.), SIV-gag (AdvancedBioscience Laboratories, Kensington, Md.), and FLT-3L (Peprotech, RockyHill, N.J.) were labeled with Alexa-Fluor 555 (Invitrogen, Carlsbad,Calif.) for direct fluorometric quantification of the amount of proteinentrapped. OVA was also encapsulated in DRVs and PLGA nanoparticles asdescribed previously^(43,44). In some experiments, ICMVs were loadedwith a recombinant vivax malaria protein (VMP) as an irrelevant antigencontrol⁴⁹ (provided by Dr. Anjali Yadava, Walter Reed Army Institute ofResearch). Capped-thiol OVA was prepared by incubating 1 mg of OVA with1.5 mM TCEP for 1 hr at RT, followed by incubation with 1.5 mMethyl-maleimide (Pierce, Rockford, Ill.) at 37° C. for 1 hr. The extentof thiol protection was >95% as assessed with Ellman's assay⁵⁰. Releaseof OVA labeled with Alexa-Fluor 555 from lipid vesicles was quantifiedin RPMI media supplemented with 10% fetal calf serum at 37° C. usingdialysis membranes with MW cutoff of 100 kDa. At regular intervals, thereleasing media were removed for quantification of fluorescence, and anequal volume of fresh media were replaced for continued monitoring ofdrug release. Residual OVA remaining at the end of the time-course wasdetermined by lipid extraction of vesicles with 1% Triton X-100treatment and measuring released protein by fluorescencespectrophotometry. OVA release assays were also performed in Hank'sbuffered saline solution supplemented with 500 ng/ml of phospholipase A(Sigma, St. Louis, Mo.). To examine stability of encapsulated cargomolecules, monoclonal rat IgG encapsulated in ICMVs was retrieved with1% triton X-100 treatment and analyzed with SDS-PAGE under non-reducingconditions with silver staining (Pierce).

Vaccination Study with ICMVs.

Groups of C57B1/6 mice (Jackson Laboratories) were immunized s.c. in thetail base with indicated doses of OVA (with or without TLR agonistMPLA). Frequencies of OVA-specific CD8⁺ T-cells and their phenotypeselicited by immunization were determined by flow cytometry analysis ofPBMCs at selected time points following staining with DAPI (todiscriminate live/dead cells), anti-CD8 alpha, anti-CD44, anti-CD62L,and SIINFEKL/H-2K^(b) peptide-MHC tetramers (Becton Dickinson). Toassess functionality of primed CD8⁺ T-cells, PBMCs were stimulated exvivo with 1 μM OVA-peptide SIINFEKL for 6 hrs with GolgiPlug (BectonDickinson), fixed, permeabilized, stained with anti-IFN-gamma and CD8alpha, and analyzed by flow cytometry. Anti-OVA IgG titers, defined asto the dilution of sera at which 450 nm OD reading is 0.5, weredetermined by ELISA analysis of sera from immunized mice. Animals werecared for following NIH, state, and local guidelines.

Statistical Analysis.

Statistical analysis was performed with Jmp 5.1 (SAS Institute Inc,Cary, N.C.). Data sets were analyzed using one- or two-way analysis ofvariance (ANOVA), followed by Tukey's HSD test for multiple comparisons.p-values less than 0.05 were considered statistically significant. Allvalues are reported as mean±s.e.m.

Results

We introduced covalent crosslinks between functionalized lipidheadgroups of adjacent, apposed bilayers within preformed MLVs to formICMVs (FIG. 2A): In a typical synthesis, dried phospholipid filmscontaining DOPC, anionic DOPG, and the anionic maleimide-headgroup lipidMPB in a 4:1:5 molar ratio were hydrated and sonicated to form simpleliposomes (step (i)). Divalent cations (e.g., Mg²⁺) were added to theliposomes to induce vesicle fusion and the formation of MLVs as reportedpreviously³² (step (ii)). To introduce crosslinks between adjacentbilayers in the MLVs, dithiolthrietol (DTT) was then added to thevesicle suspension to act as a membrane-permeable crosslinker, forming acovalent linkage between maleimide headgroups of apposed membranesbrought into proximity by the cation salt bridges formed between vesiclelayers (step (iii)). PEGylation is a well-known strategy to increase theserum stability and blood circulation half-life of lipid vesicles¹⁸.Thus as a final step, the vesicles were washed and residual maleimidegroups exposed on the external surfaces of the particles were cappedwith thiol-terminated PEG (step (iv)).

The diameter/polydispersity of the particles will determine the celltypes capable of internalizing these particles³³, while the number ofbilayers comprising the vesicle walls would be expected to impact thestability of the vesicles and their ability to retain/slowly releasecargos in the presence of serum. To evaluate these properties and betterunderstand the process of ICMV formation, we characterized the productsat each step of the synthesis: The initial liposomes formed bysonication (step (i)) had hydrodynamic diameters of ˜190 nm, and thesize increased slightly to ˜240 nm following Mg²⁺-mediated vesiclefusion (step (ii)) and subsequent DTT “stapling” of the bilayers (step(iii), Table 2). The resulting ICMVs showed a monomodal, relativelynarrow size distribution (comparable to common lipid vesicle or polymernanoparticle preparations^(12,21)), and there was no evidence for grossaggregation of particles during the crosslinking step from dynamic lightscattering (DLS) or cryoelectron microscopy (FIGS. 2A, B, to and Table2). Addition of thiol-terminated PEG to DTT-treated vesicles quenchedremaining detectable maleimide groups on the surfaces of MLVs andintroduced PEG chains on ˜2 mol % of the surface-exposed lipids of ICMVswithout significantly altering particle diameters (Table 2 and data notshown). PEGylated ICMVs stored at 4° C. or 37° C. in PBS remained stableover 7 days, and they were amenable to lyophilization with 3% sucroseadded as an excipient³⁴, highlighting their compatibility with long-termstorage conditions (Table 2 and data not shown). We imaged the initialliposomes, Mg²⁺-fused MLVs, and the final ICMVs by cryoelectronmicroscopy (FIG. 2A), and saw that crosslinking with DTT led to theformation of vesicles with thick multilamellar walls composed of tightlystacked bilayers resolved as ˜4-5 nm electron-dense striations. Themedian number of bilayers per particle was 4.4 (interquartile range[IQR], 3.3-6.9), and the median particle radius to lipid wall thicknessratio was 3.8 (IQR, 2.4-6.8) (FIGS. 2C, D). Interestingly, the majorityof ICMVs had vesicle walls composed of concentric bilayers, although afew examples of ICMVs with surface defects in the form of incompleteexternal lipid layers could also be found (data not shown). Consistentwith the increased lamellarity of the vesicles following cation-mediatedfusion and DTT crosslinking observed by electron microscopy imaging, thefraction of lipids exposed on the external surfaces of the vesiclesdecreased in steps (ii) and (iii) of the synthesis, as measured by abulk dye-quenching lamellarity assay³⁵ (Table 2). Chemical evidence forcrosslinking between the maleimide-lipids following DTT treatment wasfound in thin-layer chromatography and MALDI-TOF measurements on ICMVs(data not shown). Importantly, both the particle size and individuallamellarity distributions were monomodal, with less than 3%contaminating unilamellar vesicles and no large aggregates, which couldskew the functional properties (e.g., protein release) of the particles.The ICMVs have a size that should be avidly taken up by monocytes anddendritic cells³⁶, and a crosslinked multilamellar wall structure thatwill stabilize protein entrapment compared to traditional unilamellar ormultilamellar liposomes.

Analysis of the conditions required to form stable vesicles providedinsight into the mechanisms of ICMV formation. Followinginterbilayer-crosslinking, ICMVs could be collected by centrifuging at14,000×g for 4 min (“low-speed conditions”), whereas simple liposomes orMLVs of identical lipid composition required ultracentrifugation topellet. Using the mass of particles collected by low-speedcentrifugation as a surrogate measure of crosslinked vesicle yield, wefound that both divalent cation-mediated fusion (FIG. 2A step (ii),either Mg²⁺ or Ca²⁺) and DTT treatment (step (iii)) were required forICMV formation (Tables 1A, B). Precursor vesicles treated with eitherMg²⁺ or DTT alone even at 10× molar excess relative to maleimide groupsdid not generate significant yields of particles (Tables 1A, B). Inaddition, at least 25 mol % MPB was required to form ICMVs (Tables 1A,B); the high level of reactive headgroups required for substantial ICMVyield may reflect competition between intra-(between headgroups on thesame bilayer) and inter-bilayer crosslink formation. DTT could bereplaced with DPDPB, another membrane-permeable dithiol, but not with 2kDa PEG-dithiol under the conditions used (Tables 1A, B). ICMVs couldalso be formed using only DOPC and MPB lipids, or using cationic DOTAPin place of DOPG (data not shown). As an alternative method to formICMVs, maleimide-dithiol crosslinking could be replaced withbio-orthogonal click chemistry, employing alkyne-terminated lipids forvesicle formation and diazides for crosslinking^(37,38) (FIG. 12 anddata not shown). Thus, interbilayer-crosslinking for the formation ofstabilized vesicles is a general strategy that can be adapted to otherlipid/crosslinker chemistries. Based on their high synthetic yield andcolloidal stability, we chose to focus on PEGylated ICMVs with a lipidcomposition of DOPC:DOPG:MPB in a 4:1:5 molar ratio for further testingas protein/vaccine delivery vehicles.

To test the suitability of ICMVs for protein delivery, we examined theentrapment of several globular proteins: SIV-gag, an HIV vaccineantigen; FLT-3L, a therapeutic cytokine; and ovalbumin (OVA), a modelvaccine antigen. Protein encapsulation was achieved by rehydrating driedlipids with protein solutions in step (i) of the synthesis (FIG. 2A).The amount of encapsulated protein increased at each step of the ICMVpreparation (FIG. 4A), which may reflect additional protein entrapmentoccurring as vesicle fusion occurs in both steps (ii) and (iii). Proteinentrapment in ICMVs was not mediated by conjugation of thiols on thecargo proteins with the maleimide-functionalized lipid vesicles, as OVApre-reduced with TCEP and treated with ethyl-maleimide to block allthiol groups on the protein was encapsulated in ICMVs at levels similarto unmodified protein (76.1±6.3% vs. 83.3±8.4% for capped-thiol- vs.unmodified OVA; p=0.17). We also confirmed that disulfide linkages inmodel protein cargos were not reduced by the DTT crosslinker during thevesicle formation process and that ICMV encapsulation did not triggerprotein aggregation (data not shown). To directly compare the efficiencyand quantity of protein loading achieved with ICMVs to two of the mostcommon types of drug delivery vehicles³⁹⁻⁴¹, we compared encapsulationof OVA in liposomes, PLGA nanoparticles, and ICMVs: Using a model stablelipid composition comprised of phosphocholine, PEG-lipid, andcholesterol⁴², we formed DRVs as one of the most efficient to aqueousentrapment approaches for liposomes⁴³, and prepared OVA-loaded PLGAparticles using a double-emulsion solvent-evaporation process⁴⁴. ICMVsexhibited superior encapsulation efficiency (˜75%) compared to eitherDRVs or PLGA particles (2-, and 4-fold increases, respectively; FIG.4B), and the amount of OVA encapsulated per total particle mass (˜325 ngOVA per mg of particles) was increased in ICMVs by 1.8-, and 9-foldcompared to DRVs or PLGA particles, respectively (FIG. 4C). Thus, ICMVsappear to be effective for encapsulating a variety of globular proteins,and, at least for the model antigen OVA, ICMVs loaded protein moreefficiently than common alternative protein carriers.

We next determined whether interbilayer crosslinking enabled lipidvesicles retain biodegradability while increasing protein retention inthe presence of serum. OVA was loaded into PEGylated liposomes,Mg²⁺-fused MLVs, or ICMVs all with the same lipid composition, and thekinetics of protein release at 37° C. in media containing 10% fetal calfserum were quantified. Unilamellar liposomes quickly released theirentire payload of entrapped OVA within ˜2 days, while multilamellarMg²⁺-fused MLVs released ˜50% of their entrapped cargo over the sametime period (FIG. 4D). However, ICMVs showed a significantly enhancedretention of protein, releasing only ˜25% of their cargo by one week,and ˜90% after 30 days (FIG. 4D). Notably, ICMVs also released proteinsignificantly more slowly than unilamellar liposomes stabilized by theinclusion of cholesterol⁴² (FIG. 4D). The crosslinked vesicles alsoretained ˜95% of their entrapped protein when stored in PBS at 4° C. forover 30 days (FIG. 4E). We also examined protein release from ICMVs inconditions modeling intracellular compartments: vesicles incubated inreducing or acidic conditions for 1 day at 37° C. retained >95% ofentrapped OVA, whereas incubation with phospholipase A led to releaseof >90% OVA and rapid vesicle degradation (FIG. 4E). Thus, ICMVs exhibitenhanced stability in the presence of serum compared to traditionalliposomal formulations, but rapidly break down in the presence ofenzymes that are present within intracellular endolysosomalcompartments³¹, providing a mechanism for triggered intracellularrelease of cargo following internalization by cells. Although someprotein degradation within vesicles might be possible uponadministration in vivo prior to internalization by cells, criticaluptake and processing of antigen will occur in the first few days afterimmunization in vaccine delivery⁴⁵, when such degradation processesshould be minimal.

We hypothesized that the unique structure of ICMVs, with efficientretention of encapsulated protein antigens in the extracellularenvironment but rapid release in to endosomes/lysosomes, would provideenhanced vaccine responses. To generate vaccine ICMVs, we preparedvesicles carrying the model antigen OVA (OVA-ICMVs) and mixed thesevesicles with the molecular adjuvant monosphosphoryl lipid A (MPLA).MPLA is an FDA-approved agonist for Toll-like receptor (TLR) 4 expressedby dendritic cells, B-cells, and innate immune cells, that potentlyamplifies vaccine responses^(1,46). Antigen-loaded ICMVs mixed with MPLApromoted upregulation of costimulatory molecules on splenic andbone-marrow dendritic cells (DCs) in vitro, compared to DCs pulsed withICMVs without MPLA (FIG. 7A and data not shown). DCs pulsed with ICMVscross-presented peptides derived from OVA with greatly enhancedefficiency compared to those pulsed with soluble OVA (with or withoutadded MPLA), as determined by staining DCs with the 25-D1.16 mAb thatrecognizes the SIINFEKL peptide (OVA₂₅₇₋₂₆₄) complexed with MHC class IH-2K^(b) molecules (p<0.001, compared to soluble OVA or ICMVs-loadedwith irrelevant antigen [vivax malaria protein, VMP], FIG. 7B). SplenicDCs incubated with OVA-ICMVs+MPLA triggered robust proliferation ofOVA-specific naïve OT-1 CD8⁺ T-cells in vitro, as assessed by acarboxyfluorescein succinimidyl ester (CFSE) dilution assay. Incontrast, weak T-cell responses were detected when DCs were pulsed withequivalent doses of soluble OVA and MPLA, empty ICMVs, or VMP-ICMVs,indicating the specificity of the T-cell responses elicited by ICMVs(FIG. 7C). These results suggest that addition of MPLA allows equivalentDC activation by ICMVs or soluble OVA, but ICMVs trigger enhancedcross-presentation of the antigen, as expected for particulate antigendelivery.

To determine the influence of vesicle structure on the immune responsein vivo, we vaccinated C57B1/6 mice with equivalent doses of OVA, MPLA,and lipids (10 μg, 0.1 μg, and 142 μg, respectively) in the form ofPEGylated unilamellar liposomes, MLVs, or ICMVs. Seven days afterimmunization, we assessed the strength of the endogenous CD8⁺ T-cellresponse by analyzing the frequency of OVA peptide-MHC tetramer(antigen-specific) CD8⁺ T-cells among peripheral blood mononuclear cells(PBMCs) by flow cytometry, and found a trend toward increasing T-cellresponses in the order soluble OVA<liposomes<Mg²⁺-fused MLVs<ICMVs (FIG.9A). At 3 weeks post-immunization, Mg²⁺-fused MLVs elicited ˜100-foldgreater OVA-specific IgG titers in the sera of the animals compared tosoluble OVA or unilamellar liposomes. However, ICMV immunizationgenerated a substantially stronger humoral response, ˜1000-fold and˜10-fold greater than the soluble OVA (p<0.01) and non-crosslinked MLVimmunizations (p<0.05), respectively (FIG. 9B). Thus, the stabilizedstructure of ICMVs to promoted both T-cell and antibody responses.Enhanced T-cell and antibody responses to immunization with ICMVscompared to other formulations, could be attributed to improved antigendelivery to antigen-presenting cells (APCs), enhanced activation of DCs,enhanced antigen cross-presentation (as seen in vitro), or a combinationof these factors. To distinguish between these possibilities, mice wereimmunized with fluorophore-conjugated OVA mixed with MPLA as a soluble,liposomal, or ICMV formulation, and the draining inguinal lymph nodecells that internalized OVA were assessed on day 2. OVA delivered byICMVs was readily detected in total DCs, macrophages, and plasmacytoid(CD11c⁺B220⁺) DCs in the draining lymph nodes (dLNs), while soluble andliposomal formulations showed fluorescence barely above background(p<0.01, FIGS. 9C, D). Repeating this analysis with unlabeled OVA, wefound that administration of OVA-ICMVs with MPLA triggered a minorenhancement of co-stimulatory marker and MHC-II expression among DCs indLNs compared to soluble or liposomal OVA+MPLA (FIG. 9E and data notshown). However, using the 25-D1.16 antibody to detect OVA peptidepresentation, we readily detected OVA peptide-MHC complexes on DCs inthe dLNs following ICMV immunization, whereas soluble OVA or liposomalOVA injections did not give staining above the expected backgroundcross-reactivity of 25-D1.16 with self-peptide MHC complexes⁴⁷ (FIG.9F). All together, these results suggest that improved retention ofentrapped antigen in the crosslinked multilamellar structures of ICMVsleads to enhanced antigen delivery to APCs, followed by enhancedcross-presentation.

The multilamellar structure of ICMVs offers the opportunity to sequesternot only protein antigen (in the aqueous core) but also lipophilicmolecules (in the vesicle walls). We thus tested whether embedding MPLAthroughout the walls of the ICMVs would impact the immune response invivo, by allowing better retention of MPLA together with antigen in thevesicles. The TLR agonist was incorporated throughout the vesicle layersby co-dissolving MPLA with the other lipids in the first step of thesynthesis (int-MPLA ICMVs), and we compared these vesicles to ICMVscarrying the same amount of MPLA incorporated only on the vesiclesurfaces via a post-insertion approach (ext-MPLA ICMVs, FIG. 11A anddata not shown). Mice were immunized s.c. with OVA (10 μg) and MPLA (0.1μg or 1.0 μg) in ICMVs or soluble form, and boosted on day 21 and day 35with the same formulations. As shown in FIG. 11B, immunizations usingthe low dose of MPLA led to a barely detectable antibody responseagainst soluble OVA even following two boosts, while both int-MPLA andext-MPLA ICMVs elicited strong anti-OVA serum IgG titers by day 56. AnIgG response to soluble OVA to could be obtained using 10-fold moreMPLA, but int-MPLA ICMVs with 1.0 μg MPLA elicited higher titers thansoluble protein (˜13-fold greater on day 56). On the other hand, we didnot observe any significant level of antibodies directed against lipidcomponents of ICMVs throughout these immunization studies (data notshown).

Embedding MPLA in the multilayers of ICMVs had a more striking effect onthe CD8⁺ T-cell response to vaccination. Soluble OVA mixed with 0.1 μgMPLA elicited barely detectable antigen-specific T-cell expansion asassessed by tetramer staining on PBMCs; ext-MPLA ICMV delivery led to a2.5-fold increased tetramer⁺ T-cell population by d41 (FIG. 11C,p<0.05). Adding 10-fold more MPLA allowed soluble OVA immunizations toeventually reach T-cell responses equivalent to ext-MPLA ICMVs followingboosting. By contrast, immunization with int-MPLA ICMVs eliciteddramatically stronger CD8⁺ T-cell responses that continued to expandfollowing each boost, achieving a peak 28% tetramer⁺ T-cells in the CD8⁺T-cell population by day 41 (5-fold greater than ext-MPLA ICMVs (p<0.05)and 14-fold greater than soluble OVA+MPLA (p<0.01) FIG. 11C). Notably,int-MPLA ICMVs elicited overall a significantly higher frequency oftetramer⁺CD44⁺CD62L⁺ cells (p<0.01, FIG. 11D), a phenotype for centralmemory T-cells known to confer long-lived protection against pathogensand tumors⁴⁸. Antigen specific T-cells elicited by int-MPLA ICMVspersisted even after one month after the final boosting, with ˜11%tetramer⁺ T-cells among CD8⁺ T-cells (3-fold and 8-fold greater thanext-MPLA ICMVs and soluble OVA+MPLA, respectively, p<0.05 for both, FIG.11C). To test the functionality of T-cells expanded by theseimmunizations, we assessed the ability of CD8⁺ T-cells from peripheralblood to produce interferon-γ (IFN-γ) upon restimulation ex vivo on day49. Mice immunized with int-MPLA ICMVs had much higher levels ofIFN-γ-competent T-cells than mice receiving ext-MPLA ICMVs or solubleOVA-immunizations (p<0.05, FIG. 11E). To our knowledge, in terms of thedegree of antigen-specific T-cell expansion, persistence of memorycells, and IFN-γ functionality, this is one of the strongest endogenousT-cell responses ever reported for a protein vaccine, comparable tostrong live vectors such as recombinant viruses^(5,6). Notably, this isachieved via “homologous” boosting, repeated immunization with the sameparticle formulation, a strategy that cannot be used with many livevectors due to immune responses raised against the pathogen-baseddelivery vector itself.

These studies demonstrate the synthesis of a new class of submicronparticle reagents based on crosslinked multilamellar lipid vesicles,which combine a number of attractive features for to biomedicalapplications; the particle synthesis does not require exposure ofprotein cargos to organic solvents, the lipid basis of the particlesmakes them inherently biodegradable to metabolizable byproducts, thephospholipid shell enables modular entrapment of both lipophilic andhydrophilic cargos, proteins are encapsulated at very high levels permass of particles, and protein release from the particles can besustained over very long durations. These results suggest ICMVs may be avery effective vehicle for delivering biomacromolecules, and inparticular, for vaccine applications. The ability to achieve such strongcombined T-cell and antibody responses using a synthetic particlevaccine could open up new possibilities for vaccination in the settingof infectious disease and cancer.

REFERENCES

-   1. Guy, B. The perfect mix: recent progress in adjuvant research.    Nat Rev Microbiol 5, 505-517 (2007).-   2. Perrie, Y., Mohammed, A. R., Kirby, D. J., McNeil, S. E. &    Bramwell, V. W. Vaccine adjuvant systems: enhancing the efficacy of    sub-unit protein antigens. Int J Pharm 364, 272-280 (2008).-   3. Reed, S. G., Bertholet, S., Coler, R. N. & Friede, M. New    horizons in adjuvants for vaccine development. Trends Immunol 30,    23-32 (2009).-   4. Walker, B. D. & Burton, D. R. Toward an AIDS vaccine. Science    320, 760-764 (2008).-   5. Haglund, K., et al. Robust recall and long-term memory T-cell    responses induced by prime-boost regimens with heterologous live    viral vectors expressing human immunodeficiency virus type 1 Gag and    Env proteins. J Virol 76, 7506-7517 (2002).-   6. Flatz, L., et al. Development of replication-defective    lymphocytic choriomeningitis virus vectors for the induction of    potent CD8+ T cell immunity. Nat Med 16, 339-345 (2010).-   7. Brave, A., Ljungberg, K., Wahren, B. & Liu, M. A. Vaccine    delivery methods using viral vectors. Mol Pharm 4, 18-32 (2007).-   8. Priddy, F. H., et al. Safety and immunogenicity of a    replication-incompetent adenovirus type 5 HIV-1 Glade B gag/pol/nef    vaccine in healthy adults. Clin Infect Dis 46, 1769-1781 (2008).-   9. Hubbell, J. A., Thomas, S. N. & Swartz, M. A. Materials    engineering for immunomodulation. Nature 462, 449-460 (2009).-   10. Heath, W. R. & Carbone, F. R. Cross-presentation in viral    immunity and self-tolerance. Nat Rev Immunol 1, 126-134 (2001).-   11. Kwon, Y. J., James, E., Shastri, N. & Frechet, J. M. In vivo    targeting of dendritic cells for activation of cellular immunity    using vaccine carriers based on pH-responsive microparticles. Proc    Natl Acad Sci USA 102, 18264-18268 (2005).-   12. Hamdy, S., et al. Enhanced antigen-specific primary CD4+ and    CD8+ responses by codelivery of ovalbumin and toll-like receptor    ligand monophosphoryl lipid A in poly(D,L-lactic-co-glycolic acid)    nanoparticles. J Biomed Mater Res A 81, 652-662 (2007).-   13. Heit, A., Schmitz, F., Haas, T., Busch, D. H. & Wagner, H.    Antigen co-encapsulated with adjuvants efficiently drive protective    T cell immunity. Eur J Immunol 37, 2063-2074 (2007).-   14. Schlosser, E., et al. TLR ligands and antigen need to be    coencapsulated into the same biodegradable microsphere for the    generation of potent cytotoxic T lymphocyte responses. Vaccine 26,    1626-1637 (2008).-   15. Heffernan, M. J., Kasturi, S. P., Yang, S. C., Pulendran, B. &    Murthy, N. The stimulation of CD8+ T cells by dendritic cells pulsed    with polyketal microparticles containing ion-paired protein antigen    and poly(inosinic acid)-poly(cytidylic acid). Biomaterials 30,    910-918 (2009).-   16. Demento, S. L., et al. Inflammasome-activating nanoparticles as    modular systems for optimizing vaccine efficacy. Vaccine 27,    3013-3021 (2009).-   17. Reddy, S. T., et al. Exploiting lymphatic transport and    complement activation in nanoparticle vaccines. Nat Biotechnol 25,    1159-1164 (2007).-   18. Torchilin, V. P. Recent advances with liposomes as    pharmaceutical carriers. Nat Rev Drug Discov 4, 145-160 (2005).-   19. Gregoriadis, G., Gursel, I., Gursel, M. & McCormack, B.    Liposomes as immunological adjuvants and vaccine carriers. Journal    of Controlled Release 41, 49-56 (1996).-   20. Jeong, J. M., Chung, Y. C. & Hwang, J. H. Enhanced adjuvantic    property of polymerized liposome as compared to a phospholipid    liposome. J Biotechnol 94, 255-263 (2002).-   21. Vangala, A., et al. Comparison of vesicle based antigen delivery    systems for delivery of hepatitis B surface antigen. J Control    Release 119, 102-110 (2007).-   22. Steers, N. J., Peachman, K. K., McClain, S., Alving, C. R. &    Rao, M. Liposome-encapsulated HIV-1 Gag p24 containing lipid A    induces effector CD4+ T-cells, memory CD8+ T-cells, and    pro-inflammatory cytokines. Vaccine 27, 6939-6949 (2009).-   23. Bhowmick, S., Mazumdar, T., Sinha, R. & Ali, N. Comparison of    liposome based antigen delivery systems for protection against    Leishmania donovani. J Control Release 141, 199-207 (2010).-   24. Reddy, R., Zhou, F., Nair, S., Huang, L. & Rouse, B. T. In vivo    cytotoxic T lymphocyte induction with soluble proteins administered    in liposomes. J Immunol 148, 1585-1589 (1992).-   25. Collins, D. S., Findlay, K. & Harding, C. V. Processing of    exogenous liposome-encapsulated antigens in vivo generates class I    MHC-restricted T cell responses. J Immunol 148, 3336-3341 (1992).-   26. Wakita, D., et al. An indispensable role of type-1 IFNs for    inducing CTL-mediated complete eradication of established tumor    tissue by CpG-liposome co-encapsulated with model tumor antigen. Int    Immunol 18, 425-434 (2006).-   27. Popescu, M. C., et al. A novel proteoliposomal vaccine elicits    potent antitumor immunity in mice. Blood 109, 5407-5410 (2007).-   28. Allen, T. M., Mumbengegwi, D. R. & Charrois, G. J.    Anti-CD19-targeted liposomal doxorubicin improves the therapeutic    efficacy in murine B-cell lymphoma and ameliorates the toxicity of    liposomes with varying drug release rates. Clin Cancer Res 11,    3567-3573 (2005).-   29. Cashion, M. P. & Long, T. E. Biomimetic Design and Performance    of Polymerizable Lipids. Accounts of Chemical Research 42, 1016-1025    (2009).-   30. Hotz, J. & Meier, W. Vesicle-templated polymer hollow spheres.    Langmuir 14, 1031-1036 (1998).-   31. Mahadevan, S. & Tappel, A. L. Lysosomal lipases of rat liver and    kidney. J Biol Chem 243, 2849-2854 (1968).-   32. Papahadjopoulos, D., Nir, S. & Duzgunes, N. Molecular mechanisms    of calcium-induced membrane fusion. J Bioenerg Biomembr 22, 157-179    (1990).-   33. Zauner, W., Farrow, N. A. & Haines, A. M. In vitro uptake of    polystyrene microspheres: effect of particle size, cell line and    cell density. J Control Release 71, 39-51 (2001).-   34. Mohammed, A. R., Bramwell, V. W., Coombes, A. G. & Perrie, Y.    Lyophilisation and sterilisation of liposomal vaccines to produce    stable and sterile products. Methods 40, 30-38 (2006).-   35. Girard, P., et al. A new method for the reconstitution of    membrane proteins into giant unilamellar vesicles. Biophys J 87,    419-429 (2004).-   36. Lutsiak, M. E., Robinson, D. R., Coester, C., Kwon, G. S. &    Samuel, J. Analysis of poly(D,L-lactic-co-glycolic acid) nanosphere    uptake by human dendritic cells and macrophages in vitro. Pharm Res    19, 1480-1487 (2002).-   37. Huisgen, R. Cycloadditions—definition classification and    characterization. Angewandte Chemie-International Edition 7, 321-&    (1968).-   38. Wang, Q., et al. Bioconjugation by copper(I)-catalyzed    azide-alkyne [3+2] cycloaddition. J Am Chem Soc 125, 3192-3193    (2003).-   39. Allen, T. M. & Cullis, P. R. Drug delivery systems: entering the    mainstream. Science 303, 1818-1822 (2004).-   40. Mundargi, R. C., Babu, V. R., Rangaswamy, V., Patel, P. &    Aminabhavi, T. M. Nano/micro technologies for delivering    macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and    its derivatives. J Control Release 125, 193-209 (2008).-   41. Vasir, J. K. & Labhasetwar, V. Biodegradable nanoparticles for    cytosolic delivery of therapeutics. Adv Drug Deliv Rev 59, 718-728    (2007).-   42. Gabizon, A., et al. Prolonged circulation time and enhanced    accumulation in malignant exudates of doxorubicin encapsulated in    polyethylene-glycol coated liposomes. Cancer Res 54, 987-992 (1994).-   43. Kirby, C. & Gregoriadis, G. Dehydration-rehydration vesicles—a    simple method for high-yield drug entrapment in liposomes.    Bio-Technology 2, 979-984 (1984).-   44. Bershteyn, A., et al. Polymer-supported lipid shells, onions,    and flowers. Soft Matter 4, 1787-1791 (2008).-   45. McKee, A. S., Munks, M. W. & Marrack, P. How do adjuvants work?    Important considerations for new generation adjuvants. Immunity 27,    687-690 (2007).-   46. Mata-Haro, V., et al. The vaccine adjuvant monophosphoryl lipid    A as a TRIF-biased agonist of TLR4. Science 316, 1628-1632 (2007).-   47. Porgador, A., Yewdell, J. W., Deng, Y., Bennink, J. R. &    Germain, R. N. Localization, quantitation, and in situ detection of    specific peptide-MHC class I complexes using a monoclonal antibody.    Immunity 6, 715-726 (1997).-   48. Sallusto, F., Geginat, J. & Lanzavecchia, A. Central memory and    effector memory T cell subsets: function, generation, and    maintenance. Annu Rev Immunol 22, 745-763 (2004).-   49. Yadava, A., et al. A novel chimeric Plasmodium vivax    circumsporozoite protein induces biologically functional antibodies    that recognize both VK210 and VK247 sporozoites. Infect Immun 75,    1177-1185 (2007).-   50. Ellman, G. L. Tissue sulfhydryl groups. Arch Biochem Biophys 82,    70-77 (1959).

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin to conjunction with open-ended language such as “comprising” canrefer, in one embodiment, to A only (optionally including elements otherthan B); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is: 1-52. (canceled)
 53. A multilamellar lipid vesiclehaving at least two lipid bilayers covalently crosslinked to each otherthrough headgroups that react with covalent crosslinkers to formcovalent crosslinks between the at least two lipid bilayers, wherein themultilamellar lipid vesicle comprises an agent.
 54. The multilamellarlipid vesicle of claim 53, wherein the agent is an antibody, an antibodyfragment, a fusion protein, an enzyme, a co-factor, a receptor, aligand, an antigen, a cytokine or a chemokine.
 55. The multilamellarlipid vesicle of claim 54, wherein the agent is an antibody or anantibody fragment.
 56. The multilamellar lipid vesicle of claim 55,wherein the antibody or the antibody fragment is respectively animmunostimulatory antibody or an immunostimulatory antibody fragment.57. The multilamellar lipid vesicle of claim 55, wherein the antibody orthe antibody fragment binds to a cancer antigen.
 58. The multilamellarlipid vesicle of claim 57, wherein the cancer antigen is expressed onthe surface of a cancer cell.
 59. The multilamellar lipid vesicle ofclaim 54, wherein the agent is an antigen.
 60. The multilamellar lipidvesicle of claim 59 further comprising an adjuvant.
 61. Themultilamellar lipid vesicle of claim 59, wherein the antigen is humanpapillomavirus (HPV) protein.
 62. The multilamellar lipid vesicle ofclaim 61 further comprising an adjuvant.
 63. A method of delivering anagent to a subject comprising administering to the subject themultilamellar lipid vesicle of claim
 53. 64. A composition comprising: amultilamellar lipid vesicle having at least two lipid bilayerscovalently crosslinked to each other through headgroups that react withcovalent crosslinkers to form covalent crosslinks between the at leasttwo lipid bilayers; and an agent.
 65. The composition of claim 64,wherein the multilamellar lipid vesicle comprises the agent.
 66. Thecomposition of claim 65, wherein the agent is an antibody, an antibodyfragment, a fusion protein, an enzyme, a co-factor, a receptor, aligand, an antigen, a cytokine or a chemokine.
 67. (canceled)
 68. Thecomposition of claim 66, wherein the agent is an antibody or an antibodyfragment that binds to a cancer antigen. 69.-70. (canceled)
 71. Thecomposition of claim 66, wherein the agent is an antigen, and thecomposition further comprises an adjuvant.
 72. The composition of claim71, wherein the multilamellar lipid vesicle comprises the adjuvant. 73.The composition of claim 66, wherein the agent is an antigen that ishuman papillomavirus (HPV) protein. 74.-75. (canceled)
 76. A method ofdelivering an agent to a subject comprising administering to the subjectthe composition of claim
 64. 77. A method of delivering an agent to asubject comprising administering to a subject a multilamellar lipidvesicle having at least two lipid bilayers covalently crosslinked toeach other through headgroups that react with covalent crosslinkers toform covalent crosslinks between the at least two lipid bilayers; and anagent. 78.-80. (canceled)